Methods of selectively modulating gastrointestinal microbial growth

ABSTRACT

The present disclosure relates to methods of feeding animals by providing feed additives that modulate the gut microbiome to improve the health, nutrition, and growth performance. The present disclosure further relates to methods of modulating the microbial species present in the gastrointestinal tract of an animal. Such modulation includes, for example, modulating the level or function of microbial species.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplications No. 62/757,438 filed on Nov. 8, 2018, and U.S. ProvisionalPatent Applications No. 62/757,439 filed on Nov. 8, 2018; thedisclosures of each of which are hereby incorporated by reference intheir entirety.

BACKGROUND

Global population growth places continuous pressure on agriculture andanimal farming to improve the yield and sustainability of animalproduction. Meeting this demand requires continuous improvements to thegrowth performance of animals raised for protein. For example,increasing the feed efficiency of an animal allows the animal to achievethe same weight or productivity while consuming less feed than acomparable animal with lower feed efficiency. Since the production offeed requires the consumption of resources and energy to obtain,formulate, and transport its ingredients, improving animal growthperformance reduces the amount of resources and energy required to growthe animal. Furthermore, since feed is the largest cost of raisinganimals, improved feed efficiency provides an economic advantage to theproducer.

Feed additives have been developed to improve the body weight gain andfeed efficiency of animals. For example, antibiotic growth promoters(AGPs) saw widespread use for their ability to increase weight gain andfeed efficiency. However, increasing regulatory and consumer pressurehave moved the animal production industry away from antibiotic feedadditives, which exhibit a poor sustainability profile. In particular,non-therapeutic use of antibiotics contributes to increased microbialresistance and multi-drug-resistant strains of dangerous pathogens.

Direct fed microbials (DFMs) have been explored by the animal productionindustry as an alternative to AGPs. Unlike antibiotics, DFMs attempt tosupport the host animal by providing probiotic species that exert apositive influence on the animal's digestive system. DFMs, such ascommensal bacteria or yeasts, are generally restricted to spore-formingmicrobes so that they can withstand formulation into a dry product andincorporation into conventional feed manufacturing processes. It ishowever, challenging to formulate probiotic organisms into dry productforms without compromising the viability of the active strains. Theresulting dose variability, i.e. the number of viable organismsdelivered to the digestive track, translates to inconsistent performanceunder commercial production conditions. Furthermore, many of the mostprominent commensal gastrointestinal microbes, i.e., taxa with thegreatest beneficial impact on growth performance, are either non-sporeforming or unculturable, making it essentially impossible to form theminto a stable feed additive.

Thus, there is a need for novel feed additives and nutritionalcompositions that improve animal growth, feed efficiency, and health,including feed additives that promote growth of beneficialgastrointestinal microbes not readily formulated into DFM animal feedadditives or inhibit the growth of detrimental gastrointestinalmicrobes.

SUMMARY

In one aspect, provided herein are methods of increasing the body weightof an animal, the method comprising: administering a nutritionalcomposition comprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein the body weight of said animal is increasedrelative to the body weight of said animal prior to said administeringsaid nutritional composition to said animal, and wherein the increase inthe body weight of said animal is a larger increase relative to anincrease in body weight of a comparable control animal administered acomparable nutritional composition lacking said oligosaccharidepreparation.

In some embodiments, said body weight of said animal is at least 10 g,20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, or 100 g higher thansaid body weight of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation.

In some embodiments, said body weight of said animal is at least atleast 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, or 100 ghigher than said body weight of said animal prior to administration ofsaid nutritional composition comprising said synthetic oligosaccharidepreparation, as measured after at least 30 days, 35 days, 40 days, 45days, 50 days, 60 days, 70 days, 80 days, or 90 days after firstadministration of said nutritional composition comprising said syntheticoligosaccharide preparation, and wherein said animal ingests saidnutritional composition at least once during every twenty-four-hourperiod.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal atleast once, twice, three, four, or five times a day.

In some embodiments, said administering comprises providing saidnutritional composition to said animal to ingest at will.

In some embodiments, said animal ingests at least a portion of saidnutritional composition in over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 90, or 120 twenty-four-hour periods.

In some embodiments, said nutritional composition comprises at least 100ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900ppm, 1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. Insome embodiments, said nutritional composition comprises about 100 ppm,200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm,1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises about 500 ppmoligosaccharide preparation. In some embodiments, said nutritionalcomposition comprises from about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300ppm-600 ppm, or 300 ppm-500 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises from about 300ppm-600 ppm oligosaccharide preparation.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography.

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of decreasing the feedconversion ratio of an animal, the method comprising: administering anutritional composition comprising a base nutritional composition and asynthetic oligosaccharide preparation to an animal, wherein saidsynthetic oligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein the feed conversion ratio (FCR) of said animalis decreased relative to the FCR of said animal prior to saidadministering said nutritional composition to said animal.

In some embodiments, said feed conversion ratio (FCR) of said animal isat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% lower than said FCRof said animal prior to administration of said nutritional compositioncomprising said synthetic oligosaccharide preparation.

In some embodiments, said feed conversion ratio (FCR) of said animal isat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% lower than said FCRof said animal prior to administration of said nutritional compositioncomprising said synthetic oligosaccharide preparation, as measured afterat least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days,80 days, or 90 days after first administration of said nutritionalcomposition comprising said synthetic oligosaccharide preparation, andwherein said animal ingests said nutritional composition at least onceduring every twenty-four-hour period.

In some embodiments, the decrease in the feed conversion ratio of saidanimal is a larger decrease relative to a decrease in feed conversionratio of a comparable control animal administered a comparablenutritional composition lacking said oligosaccharide preparation.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal atleast once, twice, three, four, or five times a day.

In some embodiments, said administering comprises providing saidnutritional composition to said animal to ingest at will.

In some embodiments, said animal ingests at least a portion of saidnutritional composition in over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 90, or 120 twenty-four-hour periods.

In some embodiments, said nutritional composition comprises at least 100ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900ppm, 1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. Insome embodiments, said nutritional composition comprises about 100 ppm,200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm,1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises about 500 ppmoligosaccharide preparation. In some embodiments, said nutritionalcomposition comprises from about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300ppm-600 ppm, or 300 ppm-500 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises from about 300ppm-600 ppm oligosaccharide preparation.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography.

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of increasing the feedefficacy of an animal, the method comprising: administering anutritional composition comprising a base nutritional composition and asynthetic oligosaccharide preparation to an animal, wherein saidsynthetic oligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein the feed efficiency of said animal isincreased relative to the feed efficiency of said animal prior to saidadministering said nutritional composition to said animal.

In some embodiments, said feed efficiency of said animal is at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than said feed efficiencyof said animal prior to administration of said nutritional compositioncomprising said synthetic oligosaccharide preparation.

In some embodiments, said feed efficiency of said animal is at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than said feed efficiencyof said animal prior to administration of said nutritional compositioncomprising said synthetic oligosaccharide preparation, as measured afterat least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days,80 days, or 90 days after first administration of said nutritionalcomposition comprising said synthetic oligosaccharide preparation, andwherein said animal ingests said nutritional composition at least onceduring every twenty-four-hour period.

In some embodiments, the increase in the feed efficiency of said animalis a larger increase relative to an increase in feed efficiency of acomparable control animal administered a comparable nutritionalcomposition lacking said oligosaccharide preparation.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal atleast once, twice, three, four, or five times a day.

In some embodiments, said administering comprises providing saidnutritional composition to said animal to ingest at will.

In some embodiments, said animal ingests at least a portion of saidnutritional composition in over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 90, or 120 twenty-four-hour periods.

In some embodiments, said nutritional composition comprises at least 100ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900ppm, 1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. Insome embodiments, said nutritional composition comprises about 100 ppm,200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm,1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises about 500 ppmoligosaccharide preparation. In some embodiments, said nutritionalcomposition comprises from about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300ppm-600 ppm, or 300 ppm-500 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises from about 300ppm-600 ppm oligosaccharide preparation.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of modulating the growth ofat least one microbial species in the gastrointestinal tract of ananimal, said method comprising: administering a nutritional compositioncomprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial species in agastrointestinal sample from said animal is increased or decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from said animal prior to said administeringsaid nutritional composition to said animal.

In some embodiments, said increase or decrease in said least onemicrobial species in a gastrointestinal sample from said animal is alarger increase or decrease relative to an increase or decrease in saidat least one microbial species in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said at least one microbial species is an archaea,a bacteria, a protozoan, a virus, a bacteriophage, a parasite, or afungus. In some embodiments, said microbial species is a bacteria.

In some embodiments, said gastrointestinal sample is a biopsy of agastrointestinal tissue, a fecal sample, or a cloacal swab. In someembodiments, said gastrointestinal tissue is cecal tissue or ileumtissue. In some embodiments, said method further comprises obtainingsaid sample.

In some embodiments, said method further comprises detecting the levelof said at least one microbial species in said gastrointestinal sample.In some embodiments, said method further comprises detecting a level ofat least 2, 3, 4, 5, 6 microbial species in said gastrointestinalsample. In some embodiments, a level of at least 2, 3, 4, 5, or 6microbial species in said gastrointestinal sample from said animal areincreased or decreased relative to a level of said at least 2, 3, 4, 5,or 6 microbial species in a gastrointestinal sample from said animalprior to said administering said nutritional composition to said animal.In some embodiments, a level of at least 2, 3, 4, 5, or 6 microbialspecies in a gastrointestinal sample from said animal are increased ordecreased relative to a level of said at least 2, 3, 4, 5, or 6, or moremicrobial species in a gastrointestinal sample from a control animalthat has been administered a comparable nutritional composition lackingsaid synthetic oligosaccharide preparation.

In some embodiments, said method comprises promoting the growth of saidat least one microbial species, and wherein said level of at least onemicrobial species in said gastrointestinal sample is increased relativeto a level of said at least one microbial species in a gastrointestinalsample from said animal prior to said administering said nutritionalcomposition to said animal. In some embodiments, said increase in saidleast one microbial species in a gastrointestinal sample from saidanimal is a larger increase relative to an increase in said at least onemicrobial species in a gastrointestinal sample from a comparable controlanimal that has been administered a comparable nutritional compositionlacking said synthetic oligosaccharide preparation.

In some embodiments, said method comprises promoting the growth of saidat least one microbial species, and wherein said level of at least onemicrobial species in said gastrointestinal sample is increased relativeto a level of said at least one microbial species in a gastrointestinalsample from a comparable control animal that has been administered acomparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial species is beneficial to the healthof the animal. In some embodiments, said microbial species is beneficialto the gastrointestinal health of the animal. In some embodiments, saidmicrobial species is non-pathogenic to said animal. In some embodiments,said microbial species is non-pathogenic to humans.

In some embodiments, said microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, said microbial species is selectedfrom the group consisting of: Bacteroides clarus, Bacteroides dorei,Odoribacter splanchinicus, and Barnesiella intestinihominis. In someembodiments, said microbial species is selected from Group 1 as listedin Table 26. In some embodiments, said microbial species is selectedfrom Group 2 as listed in Table 26. In some embodiments, said microbialspecies is selected from Group 3 as listed in Table 26.

In some embodiments, each of said at least 2, 3, 4, 5, or 6 microbialspecies improve the health of the animal. In some embodiments, each ofsaid at least 2, 3, 4, 5, or 6 microbial species improve thegastrointestinal health of the animal. In some embodiments, each of saidat least 2, 3, 4, 5, or 6 microbial species are non-pathogenic to saidanimal. In some embodiments, each of said at least 2, 3, 4, 5, or 6microbial species are non-pathogenic to humans. In some embodiments,each of said at least 2, 3, 4, 5, or 6 microbial species in saidgastrointestinal sample from said animal are increased relative to alevel of said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from said animal prior to administration of saidnutritional composition to said animal. In some embodiments, each ofsaid at least 2, 3, 4, 5, or 6 microbial species in a gastrointestinalsample from said animal are increased relative to a level of said atleast 2, 3, 4, 5, or 6 microbial species in a gastrointestinal samplefrom a control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation. In some embodiments, at least one of said at least 2, 3, 4,5, or 6 microbial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, at least one of said at least 2, 3, 4, 5, or 6 microbialspecies is selected from the group consisting of: Bacteroides clarus,Bacteroides dorei, Odoribacter splanchinicus, and Barnesiellaintestinihominis. In some embodiments, said gastrointestinal samplecomprises at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, said gastrointestinal sample comprises at least0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of at least one ofBacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, said method comprises inhibiting the growth thegrowth of said at least one microbial species, and wherein said level ofat least one microbial species in a gastrointestinal sample is decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from said animal prior to said administeringsaid nutritional composition to said animal. In some embodiments, saiddecrease in said least one microbial species in a gastrointestinalsample from said animal is a larger decrease relative to an increase insaid at least one microbial species in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said method comprises inhibiting the growth thegrowth of said at least one microbial species, and wherein said level ofat least one microbial species in a gastrointestinal sample is decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from a comparable control animal that has beenadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial species is detrimental to the healthof said animal. In some embodiments, said microbial species isdetrimental to the gastrointestinal health of said animal. In someembodiments, said microbial species is pathogenic to said animal. Insome embodiments, said microbial species is pathogenic to humans but notpathogenic to said animal.

In some embodiments, said microbial species is Helicobacter pullorum. Insome embodiments, said microbial species belongs to the phylumProteobacteria. In some embodiments, said microbial species belongs tothe genus Helicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. Insome embodiments, said microbial species is selected from the groupconsisting of: Helicobacter pullorum, Proteobacteria johnsonii,Escherichia coli, Camplobacter jejuni, and Lactobacillus crispatus. Insome embodiments, said microbial species is selected from Group 2 aslisted in Table 26. In some embodiments, said microbial species isselected from Group 3 as listed in Table 26. In some embodiments, saidmicrobial species is selected from Group 4 as listed in Table 26.

In some embodiments, each of said at least 2, 3, 4, 5, or 6 microbialspecies is detrimental to the health of the animal. In some embodiments,each of said at least 2, 3, 4, 5, or 6 microbial species in saidgastrointestinal sample from said animal is decreased relative to alevel of said at least 2, 3, 4, 5, or 6, or more microbial species in agastrointestinal sample from said animal prior to said administeringsaid nutritional composition to said animal. In some embodiments, eachof said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from said animal are decreased relative to alevel of said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from a control animal that has been administereda comparable nutritional composition lacking said syntheticoligosaccharide preparation. In some embodiments, at least one of saidat least 2, 3, 4, 5, or 6 microbial species belongs to the phylumProteobacteria. In some embodiments, at least one of said at least 2, 3,4, 5, or 6 microbial species belongs to the genus Helicobacter,Escherichia, Salmonella, Vibrio, or Yersinia. In some embodiments, atleast one of said at least 2, 3, 4, 5, or 6 microbial species isselected from the group consisting of: Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Camplobacter jejuni, andLactobacillus crispatus. In some embodiments, said gastrointestinalsample comprises less than 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10% of at least one microbial species that belongs to the genusHelicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. In someembodiments, said gastrointestinal sample comprises at less than 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of at least one ofHelicobacter pullorum, Proteobacteria johnsonii, Escherichia coli,Camplobacter jejuni, and Lactobacillus crispatus.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal atleast once, twice, three, four, or five times a day.

The method of any preceding claim, wherein said administering comprisesproviding the nutritional composition to said animal to ingest at will.

In some embodiments, said animal ingests at least a portion of saidnutritional composition in over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 90, or 120 twenty-four-hour periods.

In some embodiments, said nutritional composition comprises at least 100ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900ppm, 1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. Insome embodiments, said nutritional composition comprises about 100 ppm,200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm,1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises about 500 ppmoligosaccharide preparation. In some embodiments, said nutritionalcomposition comprises from about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300ppm-600 ppm, or 300 ppm-500 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises from about 300ppm-600 ppm oligosaccharide preparation.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of modulating the growth ofat least one microbial species in the gastrointestinal tract of ananimal, said method comprising: administering a nutritional compositioncomprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial species in agastrointestinal sample from said animal is increased or decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from a comparable control animal that has beenadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said at least one microbial species is an archaea,a bacteria, a protozoan, a virus, a bacteriophage, a parasite, or afungus. In some embodiments, said microbial species is a bacteria.

In some embodiments, said gastrointestinal sample is a biopsy of agastrointestinal tissue, a fecal sample, or a cloacal swab. In someembodiments, said gastrointestinal tissue is cecal tissue or ileumtissue. In some embodiments, said method further comprises obtainingsaid sample.

In some embodiments, said method further comprises detecting the levelof said at least one microbial species in said gastrointestinal sample.In some embodiments, said method further comprises detecting a level ofat least 2, 3, 4, 5, 6 microbial species in said gastrointestinalsample. In some embodiments, a level of at least 2, 3, 4, 5, or 6microbial species in said gastrointestinal sample from said animal areincreased or decreased relative to a level of said at least 2, 3, 4, 5,or 6 microbial species in a gastrointestinal sample from said animalprior to said administering said nutritional composition to said animal.In some embodiments, a level of at least 2, 3, 4, 5, or 6 microbialspecies in a gastrointestinal sample from said animal are increased ordecreased relative to a level of said at least 2, 3, 4, 5, or 6, or moremicrobial species in a gastrointestinal sample from a control animalthat has been administered a comparable nutritional composition lackingsaid synthetic oligosaccharide preparation.

In some embodiments, said method comprises promoting the growth of saidat least one microbial species, and wherein said level of at least onemicrobial species in said gastrointestinal sample is increased relativeto a level of said at least one microbial species in a gastrointestinalsample from said animal prior to said administering said nutritionalcomposition to said animal. In some embodiments, said increase in saidleast one microbial species in a gastrointestinal sample from saidanimal is a larger increase relative to an increase in said at least onemicrobial species in a gastrointestinal sample from a comparable controlanimal that has been administered a comparable nutritional compositionlacking said synthetic oligosaccharide preparation.

In some embodiments, said method comprises promoting the growth of saidat least one microbial species, and wherein said level of at least onemicrobial species in said gastrointestinal sample is increased relativeto a level of said at least one microbial species in a gastrointestinalsample from a comparable control animal that has been administered acomparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial species is beneficial to the healthof the animal. In some embodiments, said microbial species is beneficialto the gastrointestinal health of the animal. In some embodiments, saidmicrobial species is non-pathogenic to said animal. In some embodiments,said microbial species is non-pathogenic to humans.

In some embodiments, said microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, said microbial species is selectedfrom the group consisting of: Bacteroides clarus, Bacteroides dorei,Odoribacter splanchinicus, and Barnesiella intestinihominis. In someembodiments, said microbial species is selected from Group 1 as listedin Table 26. In some embodiments, said microbial species is selectedfrom Group 2 as listed in Table 26. In some embodiments, said microbialspecies is selected from Group 3 as listed in Table 26.

In some embodiments, each of said at least 2, 3, 4, 5, or 6 microbialspecies improve the health of the animal. In some embodiments, each ofsaid at least 2, 3, 4, 5, or 6 microbial species improve thegastrointestinal health of the animal. In some embodiments, each of saidat least 2, 3, 4, 5, or 6 microbial species are non-pathogenic to saidanimal. In some embodiments, each of said at least 2, 3, 4, 5, or 6microbial species are non-pathogenic to humans. In some embodiments,each of said at least 2, 3, 4, 5, or 6 microbial species in saidgastrointestinal sample from said animal are increased relative to alevel of said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from said animal prior to administration of saidnutritional composition to said animal. In some embodiments, each ofsaid at least 2, 3, 4, 5, or 6 microbial species in a gastrointestinalsample from said animal are increased relative to a level of said atleast 2, 3, 4, 5, or 6 microbial species in a gastrointestinal samplefrom a control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation. In some embodiments, at least one of said at least 2, 3, 4,5, or 6 microbial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, at least one of said at least 2, 3, 4, 5, or 6 microbialspecies is selected from the group consisting of: Bacteroides clarus,Bacteroides dorei, Odoribacter splanchinicus, and Barnesiellaintestinihominis. In some embodiments, said gastrointestinal samplecomprises at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, said gastrointestinal sample comprises at least0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of at least one ofBacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, said method comprises inhibiting the growth thegrowth of said at least one microbial species, and wherein said level ofat least one microbial species in a gastrointestinal sample is decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from said animal prior to said administeringsaid nutritional composition to said animal. In some embodiments, saiddecrease in said least one microbial species in a gastrointestinalsample from said animal is a larger decrease relative to an increase insaid at least one microbial species in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said method comprises inhibiting the growth thegrowth of said at least one microbial species, and wherein said level ofat least one microbial species in a gastrointestinal sample is decreasedrelative to a level of said at least one microbial species in agastrointestinal sample from a comparable control animal that has beenadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial species is detrimental to the healthof said animal. In some embodiments, said microbial species isdetrimental to the gastrointestinal health of said animal. In someembodiments, said microbial species is pathogenic to said animal. Insome embodiments, said microbial species is pathogenic to humans but notpathogenic to said animal.

In some embodiments, said microbial species is Helicobacter pullorum. Insome embodiments, said microbial species belongs to the phylumProteobacteria. In some embodiments, said microbial species belongs tothe genus Helicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. Insome embodiments, said microbial species is selected from the groupconsisting of: Helicobacter pullorum, Proteobacteria johnsonii,Escherichia coli, Camplobacter jejuni, and Lactobacillus crispatus. Insome embodiments, said microbial species is selected from Group 2 aslisted in Table 26. In some embodiments, said microbial species isselected from Group 3 as listed in Table 26. In some embodiments, saidmicrobial species is selected from Group 4 as listed in Table 26.

In some embodiments, each of said at least 2, 3, 4, 5, or 6 microbialspecies is detrimental to the health of the animal. In some embodiments,each of said at least 2, 3, 4, 5, or 6 microbial species in saidgastrointestinal sample from said animal is decreased relative to alevel of said at least 2, 3, 4, 5, or 6, or more microbial species in agastrointestinal sample from said animal prior to said administeringsaid nutritional composition to said animal. In some embodiments, eachof said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from said animal are decreased relative to alevel of said at least 2, 3, 4, 5, or 6 microbial species in agastrointestinal sample from a control animal that has been administereda comparable nutritional composition lacking said syntheticoligosaccharide preparation. In some embodiments, at least one of saidat least 2, 3, 4, 5, or 6 microbial species belongs to the phylumProteobacteria. In some embodiments, at least one of said at least 2, 3,4, 5, or 6 microbial species belongs to the genus Helicobacter,Escherichia, Salmonella, Vibrio, or Yersinia. In some embodiments, atleast one of said at least 2, 3, 4, 5, or 6 microbial species isselected from the group consisting of: Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Camplobacter jejuni, andLactobacillus crispatus. In some embodiments, said gastrointestinalsample comprises less than 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10% of at least one microbial species that belongs to the genusHelicobacter, Escherichia, Salmonella, Vibrio, or Yersinia. In someembodiments, said gastrointestinal sample comprises at less than 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% of at least one ofHelicobacter pullorum, Proteobacteria johnsonii, Escherichia coli,Camplobacter jejuni, and Lactobacillus crispatus.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, said nutritional composition comprising saidsynthetic oligosaccharide preparation is administered to said animal atleast once, twice, three, four, or five times a day.

The method of any preceding claim, wherein said administering comprisesproviding the nutritional composition to said animal to ingest at will.

In some embodiments, said animal ingests at least a portion of saidnutritional composition in over at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,20, 30, 40, 50, 60, 90, or 120 twenty-four-hour periods.

In some embodiments, said nutritional composition comprises at least 100ppm, 200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900ppm, 1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. Insome embodiments, said nutritional composition comprises about 100 ppm,200 ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm,1000 ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises about 500 ppmoligosaccharide preparation. In some embodiments, said nutritionalcomposition comprises from about 100 ppm-2000 ppm, 100 ppm-1500 ppm, 100ppm-1000 ppm, 100 ppm-900 ppm, 100 ppm-800 ppm, 100 ppm-700 ppm, 100ppm-600 ppm, 100 ppm-500 ppm, 100 ppm-400 ppm, 100 ppm-300 ppm, 100ppm-200 ppm, 200 ppm-1000 ppm, 200 ppm-800 ppm, 200 ppm-700 ppm, 200ppm-600 ppm, 200 ppm-500 ppm, 300 ppm-1000 ppm, 300 ppm-700 ppm, 300ppm-600 ppm, or 300 ppm-500 ppm oligosaccharide preparation. In someembodiments, said nutritional composition comprises from about 300ppm-600 ppm oligosaccharide preparation.

In some embodiments, said animal has an increased body weight relativeto a body weight of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation. In some embodiments, said body weight of said animal is atleast 1%, 2%, 3%, 4%, 5%, 5%, 7%, 8%, 9%, or 10% increased relative tosaid body weight of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation. In some embodiments, said increase in body weight is alarger increase relative to a comparable control animal administered acomparable nutritional composition lacking said syntheticoligosaccharide preparation. In some embodiments, said body weight ofsaid animal is at least 1%, 2%, 3%, 4%, 5%, 5%, 7%, 8%, 9%, or 10%increased relative to said body weight of said comparable control animaladministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said animal has an increased feed efficiencyrelative to a feed efficiency of said animal prior to administration ofsaid nutritional composition comprising said synthetic oligosaccharidepreparation. In some embodiments, said feed efficiency of said animal isat least 1%, 2%, 3%, 4%, 5%, 5%, 7%, 8%, 9%, or 10% increased relativeto said feed efficiency of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation. In some embodiments, said animal has said increase in feedefficiency is a larger increase relative to a comparable control animaladministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation. In some embodiments, said increase in feedefficiency of said animal is at least 1%, 2%, 3%, 4%, 5%, 5%, 7%, 8%,9%, or 10% increased relative to said body weight of said comparablecontrol animal administered a comparable nutritional composition lackingsaid synthetic oligosaccharide preparation.

In some embodiments, said animal has a decreased feed conversion ratio(FCR) relative to an FCR of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation. In some embodiments, said feed conversion ratio of saidanimal is at least 1%, 2%, 3%, 4%, 5%, 5%, 7%, 8%, 9%, or 10% decreasedrelative to the feed conversion ratio of said animal prior toadministration of said nutritional composition comprising said syntheticoligosaccharide preparation. In some embodiments, said animal has saiddecrease in feed conversion ratio is a larger decrease relative to acomparable control animal administered a comparable nutritionalcomposition lacking said synthetic oligosaccharide preparation. In someembodiments, said feed conversion ratio of said animal is at least 1%,2%, 3%, 4%, 5%, 5%, 7%, 8%, 9%, or 10% decreased relative to the feedconversion ratio of said comparable control animal administered acomparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, a life expectancy or survival rate of said animalis increased relative to a comparable control animal that wasadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said level of said at least one microbial speciesis determined by DNA sequencing or RNA sequencing. In some embodiments,said level of said at least one microbial species is determined byshotgun DNA sequencing.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of modulating expression ofat least one microbial protein within the gastrointestinal tract of ananimal, said method comprising: administering a nutritional compositioncomprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial protein in agastrointestinal sample is increased or decreased relative to a level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal.

In some embodiments, said increase or decrease in said least onemicrobial protein in a gastrointestinal sample from said animal is alarger increase or decrease relative to an increase or decrease in saidat least one microbial protein in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is decreased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is decreased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is increased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal. In some embodiments, said level of said at least onemicrobial protein in said gastrointestinal sample is increased relativeto said level of said at least one microbial protein in agastrointestinal sample from a comparable control animal that has beenadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial protein is a hydrolytic enzyme, aprotein involved in digestion (e.g. hydrolytic enzymatic digestion), ora protein involved in metabolism.

In some embodiments, said at least one microbial protein is an archaeaprotein, a bacterial protein, a protozoan protein, a viral protein, abacteriophage protein, a parasite protein, or a fungal protein. In someembodiments, said microbial species is a bacterial protein.

In some embodiments, said gastrointestinal sample is a biopsy of agastrointestinal tissue or a fecal sample. In some embodiments, saidgastrointestinal tissue is cecal tissue or ileum tissue.

In some embodiments, said method further comprises obtaining saidgastrointestinal sample. In some embodiments, said method furthercomprises detecting the level of said at least one microbial protein insaid gastrointestinal sample.

In some embodiments, said at least one microbial protein is acarbohydrate active enzyme (CAZymes). In some embodiments, said at leastone microbial protein is a sus-like polysaccharide utilization loci(PULs) protein. In some embodiments, said at least one microbial proteinis selected from the group consisting of: GH127 (CAZymeα-L-arabinofuranosidase), susC (outer membrane protein involved instarch binding) and susD (starch binding protein).

In some embodiments, said level of said at least one microbial proteinis determined by analyzing RNA or protein expression.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In one aspect, provided herein are methods of modulating expression ofat least one microbial protein within the gastrointestinal tract of ananimal, the method comprising: administering a nutritional compositioncomprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial protein in agastrointestinal sample is increased or decreased relative to a level ofsaid at least one microbial protein in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is decreased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is decreased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.

In some embodiments, said level of said at least one microbial proteinin said gastrointestinal sample is increased relative to said level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal. In some embodiments, said level of said at least onemicrobial protein in said gastrointestinal sample is increased relativeto said level of said at least one microbial protein in agastrointestinal sample from a comparable control animal that has beenadministered a comparable nutritional composition lacking said syntheticoligosaccharide preparation.

In some embodiments, said microbial protein is a hydrolytic enzyme, aprotein involved in digestion (e.g. hydrolytic enzymatic digestion), ora protein involved in metabolism.

In some embodiments, said at least one microbial protein is an archaeaprotein, a bacterial protein, a protozoan protein, a viral protein, abacteriophage protein, a parasite protein, or a fungal protein. In someembodiments, said microbial species is a bacterial protein.

In some embodiments, said gastrointestinal sample is a biopsy of agastrointestinal tissue or a fecal sample. In some embodiments, saidgastrointestinal tissue is cecal tissue or ileum tissue.

In some embodiments, said method further comprises obtaining saidgastrointestinal sample. In some embodiments, said method furthercomprises detecting the level of said at least one microbial protein insaid gastrointestinal sample.

In some embodiments, said at least one microbial protein is acarbohydrate active enzyme (CAZymes). In some embodiments, said at leastone microbial protein is a sus-like polysaccharide utilization loci(PULs) protein. In some embodiments, said at least one microbial proteinis selected from the group consisting of: GH127 (CAZymeα-L-arabinofuranosidase), susC (outer membrane protein involved instarch binding) and susD (starch binding protein).

In some embodiments, said level of said at least one microbial proteinis determined by analyzing RNA or protein expression.

In some embodiments, said animal is a poultry, seafood, sheep, cow,cattle, buffalo, bison, pig, cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, fish, orbird. In some embodiments, said animal is poultry. In some embodiments,said animal is a chicken, turkey, duck, or goose. In some embodiments,said chicken is a broiler chicken, a layer chicken, or a breederchicken. In some embodiments, said animal is a pig. In some embodiments,said pig is a nursery pig, a grower pig, or a finisher pig. In someembodiments, said animal is a fish. In some embodiments, said fish is asalmon, a tilapia, or a tropical fish.

In some embodiments, said nutritional composition is an animal feedcomposition. In some embodiments, said base nutritional composition isbase animal feed.

In some embodiments, said relative abundance is determined by LC-MS/MS.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions decreases monotonically with itsdegree of polymerization. In some embodiments, said relative abundanceof oligosaccharides in each of the n fractions decreases monotonicallywith its degree of polymerization.

In some embodiments, n is at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In some embodiments, said DP2 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises from about 5% to about10% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP2 fraction comprises from about1% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP2 fraction comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP2fraction comprises from about 2% to about 12% of anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, said DP1 fraction comprises from about2% to about 12% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP1 fraction comprisesfrom about 1% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP1fraction comprises from about 0.5% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP1 fraction comprises from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises less than 15%, lessthan 12%, less than 11%, less than 10%, less than 9%, less than 8%, lessthan 7%, less than 6%, less than 5%, less than 4%, less than 3%, lessthan 2%, or less than 1% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said DP3 fraction comprisesfrom about 2% to about 12% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, said DP3fraction comprises from about 1% to about 10% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said DP3 fraction comprises from about 0.5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said DP3 fraction comprises from about 5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises fromabout 2% to about 12% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, said oligosaccharidepreparation comprises from about 0.5% to about 10% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,said oligosaccharide preparation comprises from about 1% to about 10%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises from about5% to about 10% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, said DP2 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP1 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said DP3 fraction comprises greater than 0.6%,greater than 0.8%, greater than 1.0%, greater than 1.5%, greater than2%, greater than 3%, greater than 4%, greater than 5%, greater than 6%,greater than 7%, greater than 8%, greater than 9%, greater than 10%,greater than 11%, or greater than 12% anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation comprises greaterthan 0.5%, 0.6%, greater than 0.8%, greater than 1.0%, greater than1.5%, greater than 2%, greater than 3%, greater than 4%, greater than5%, greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, or greater than 12% anhydro-subunitcontaining oligosaccharides by relative abundance.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent of from about 1% to about 40% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP2 fractioncontent of from about 1% to about 35% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP3 fractioncontent of from about 1% to about 30% by weight as determined by liquidchromatography.

In some embodiments, said oligosaccharide preparation has a DP4 fractioncontent of from about 0.1% to about 20% by weight as determined byliquid chromatography

In some embodiments, said oligosaccharide preparation has a DP5 fractioncontent of from about 0.1% to about 15% by weight as determined byliquid chromatography.

In some embodiments, a ratio of the DP2 fraction to the DP1 fraction isfrom about 0.02 to about 0.40 as determined by liquid chromatography.

In some embodiments, a ratio of the DP3 fraction to the DP2 fraction isfrom about 0.01 to about 0.30 as determined by liquid chromatography.

In some embodiments, an aggregate content of the DP1 and the DP2fractions in the oligosaccharide preparation is less than 50%, less than40%, or less than 30% as determined by liquid chromatography.

In some embodiments, said oligosaccharide preparation comprises at least103, at least 104, at least 105, at least 106 or at least 109 differentoligosaccharide species.

In some embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits.

In some embodiments, each of said anhydro-subunit containingoligosaccharides comprises one or more anhydro-subunits that areproducts of thermal dehydration of monosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-subunits selected from anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, and anhydro-xylose.

In some embodiments, said oligosaccharide preparation comprises one ormore anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunits.

In some embodiments, said DP1 fraction comprises1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, said DP1 fraction comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits.

In some embodiments, a ratio of the 1,6-anhydro-β-D-glucofuranose to the1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, from about 9:1to about 1:10, from about 8:1 to about 1:10, from about 7:1 to about1:10, from about 6:1 to about 1:10, from about 5:1 to about 1:10, fromabout 4:1 to about 1:10, from about 3:1 to about 1:10, from about 2:1 toabout 1:10, from about 10:1 to about 1:9, from about 10:1 to about 1:8,from about 10:1 to about 1:7, from about 10:1 to about 1:6, from about10:1 to about 1:5, from about 10:1 to about 1:4, from about 10:1 toabout 1:3, from about 10:1 to about 1:2, or from about 1:1 to about 3:1in the oligosaccharide reparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4,about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10 inthe oligosaccharide preparation. In some embodiments, a ratio of the1,6-anhydro-β-D-glucofuranose to the 1,6-anhydro-β-D-glucopyranose isabout 2:1 in the oligosaccharide preparation.

In some embodiments, said DP2 fraction comprises at least 5 species ofanhydro-subunit containing oligosaccharides. In some embodiments, saidDP2 fraction comprises about 5 to 10 species of anhydro-subunitcontaining oligosaccharides.

In some embodiments, said oligosaccharide preparation comprises one ormore sugar caramelization products. In some embodiments, said sugarcaramelization products are selected from a group consisting of:methanol; ethanol; furan; methyl glyoxal; 2-methyl furan; vinyl acetate;glycolaldehyde; acetic acid; acetol; furfural; 2-furanmethanol;3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one; 5-methyl furfural;2(5H)-furanone; 2 methyl cyclopentenolone; levoglucosenone; cyclichydroxyl lactone; 1,4,3,6-dianhydro-α-D-glucopyranose; dianhydroglucopyranose; and 5-hydroxy methyl furfural (5-hmf).

In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, 95%, or 99%of the anhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 300 to about 5000 g/mol asdetermined by high-performance liquid chromatography (HPLC). In someembodiments, said oligosaccharide preparation has a weight averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a weight average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC. In some embodiments, saidoligosaccharide preparation has a number average molecular weight offrom about 300 to about 5000 g/mol as determined by HPLC. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 300 to about 2500 g/mol as determined byHPLC. In some embodiments, said oligosaccharide preparation has a numberaverage molecular weight of from about 500 to about 2000 g/mol asdetermined by HPLC. In some embodiments, said oligosaccharidepreparation has a number average molecular weight of from about 500 toabout 1500 g/mol as determined by HPLC.

In some embodiments, said oligosaccharide preparation has a weightaverage molecular weight of from about 2000 to about 2800 g/mol. In someembodiments, said oligosaccharide preparation has a number averagemolecular weight of from about 1000 to about 2000 g/mol.

In some embodiments, said relative abundance of oligosaccharides in atleast 5, 10, 20, or 30 DP fractions of said oligosaccharide preparationdecreases monotonically with its degree of polymerization. In someembodiments, the relative abundance of oligosaccharides in each of the nfractions of said oligosaccharide preparation decreases monotonicallywith its degree of polymerization. In some embodiments, n is at least 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 100.

In some embodiments, at least one fraction of said oligosaccharidepreparation comprises less than 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%,18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,or 2% anhydro-subunit containing oligosaccharides by relative abundance.In some embodiments, said oligosaccharide preparation comprises lessthan 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%,13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,each fraction of said oligosaccharide preparation comprises less than80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%,12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,at least one fraction of said oligosaccharide preparation comprises lessthan 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, saidoligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, or 2% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, each fraction of said oligosaccharidepreparation comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, at least one fraction of said oligosaccharidecomprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, said oligosaccharide preparation comprises greaterthan 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%,17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,each fraction of said oligosaccharide preparation comprises greater than2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,at least one fraction of said oligosaccharide preparation comprisesgreater than 20%, 21%, 22%, 23%, 24%, or 25% anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, saidoligosaccharide preparation comprises greater than 20%, 21%, 22%, 23%,24%, or 25% anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, each fraction of said oligosaccharidepreparation comprises greater than 20%, 21%, 22%, 23%, 24%, or 25%anhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, more than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%,55%, 50%, 45%, 40%, 35%, or 30% of the anhydro-subunit containingoligosaccharides have only one anhydro-subunit.

In some embodiments, said oligosaccharide preparation has a DP1 fractioncontent from 1 to 40% by relative abundance. In some embodiments, saidoligosaccharide preparation has a DP2 fraction content from 1 to 35% byrelative abundance. In some embodiments, said oligosaccharidepreparation has a DP3 fraction content from 1 to 30% by relativeabundance. In some embodiments, said oligosaccharide preparation has aDP4 fraction content from 0.1 to 20% by relative abundance. In someembodiments, said oligosaccharide preparation comprises a DP5 fractioncontent from 0.1 to 15% by relative abundance. In some embodiments, saidoligosaccharide preparation comprises a DP2 fraction and a DP1 fraction,wherein the ratio of said DP2 fraction to said DP1 fraction is 0.02-0.40by relative abundance. In some embodiments, said oligosaccharidepreparation comprises a DP3 fraction and a DP2 fraction, wherein theratio of said DP3 fraction to said DP2 fraction in said oligosaccharidepreparation is 0.01-0.30 by relative abundance. In some embodiments,said oligosaccharide preparation comprises a DP1 fraction and a DP2fraction, wherein the aggregate content of said DP1 and said DP2fractions in said oligosaccharide preparation is less than 50, 30, or10% by relative abundance. In some embodiments, said oligosaccharidepreparation comprises at least 25, 50, 75, 100, 103, 104, 105, 106, 109,110, 120, 150, or 200 different oligosaccharide species.

In some embodiments, at least two independent oligosaccharides of saidoligosaccharide preparation comprise different anhydro-subunits. In someembodiments, said oligosaccharide preparation comprises at least oneanhydro-subunit that is a product of reversible thermal dehydration of amonosaccharide. In some embodiments, said oligosaccharide preparationcomprises at least one anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, or anhydro-xylosesubunit. In some embodiments, said oligosaccharide preparation comprisesat least one anhydro-glucose, anhydro-galactose, anhydro-mannose, oranhydro-fructose subunit.

In some embodiments, said oligosaccharide preparation comprises at leastone1,6-anhydro-β-D-glucofuranose or 1,6-anhydro-β-D-glucopyranosesubunit. In some embodiments, said oligosaccharide preparation comprisesat least one 1,6-anhydro-β-D-glucofuranose subunit and at least one1,6-anhydro-β-D-glucopyranose anhydro-subunit. In some embodiments, aratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranosein said oligosaccharide preparation is from about 10:1 to 1:10, 9:1 to1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10,3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1.In some embodiments, a ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose in said oligosaccharide preparation isabout 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4,1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10. In some embodiments, a ratio of1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose in saidoligosaccharide preparation is about 2:1. In some embodiments, the ratioof 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose isabout from 10:1 to 1:10, 9:1 to 1:10, 8:1 to 1:10, 7:1 to 1:10, 6:1 to1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to 1:10, 2:1 to 1:10, 10:1 to 1:9,10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6, 10:1 to 1:5, 10:1 to 1:4, 10:1 to1:3, 10:1 to 1:2, or 1:1 to 3:1 in each fraction. In some embodiments, aratio of 1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranoseis about 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3,1:4, 1:5, 1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in each fraction of saidoligosaccharide preparation. In some embodiments, a ratio of1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about2:1 in each fraction of said oligosaccharide preparation. In someembodiments, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or90% of said anhydro-subunits in said oligosaccharide preparation areselected from a group consisting of 1,6-anhydro-β-D-glucofuranose and1,6-anhydro-β-D-glucopyranose.

In some embodiments, said oligosaccharide preparation comprises at leastone anhydro-subunit that is a sugar caramelization product. In someembodiments, said sugar caramelization product is selected from a groupconsisting of: methanol; ethanol; furan; methyl glyoxal; 2-methyl furan;vinyl acetate; glycolaldehyde; acetic acid; acetol; furfural;2-furanmethanol; 3-furanmethanol; 2-hydroxy cyclopent-2-en-1-one;5-methyl furfural; 2(5H)-furanone; 2 methyl cyclopentenolone;levoglucosenone; cyclic hydroxyl lactone;1,4,3,6-dianhydro-α-D-glucopyranose; dianhydro glucopyranose; and5-hydroxy methyl furfural (5-hmf). In some embodiments, from about 0.1%to 5%, 0.1% to 2%, or 0.1% to 1% of said anhydro-subunits in saidoligosaccharide preparation are caramelization products.

In some embodiments, at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or99% of the anhydro-subunit containing oligosaccharides in saidoligosaccharide preparation comprise a chain-end anhydro-subunit.

In some embodiments, the weight average molecular weight of saidoligosaccharide preparation is from about 300 to 5000 g/mol, 500 to 5000g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1300 g/mol, 400to 1200 g/mol, 400 to 1100 g/mol, 500 to 1300 g/mol, 500 to 1200 g/mol,500 to 1100 g/mol, 600 to 1300 g/mol, 600 to 1200 g/mol, or 600 to 1100g/mol. In some embodiments, the number average molecular weight of saidoligosaccharide preparation is from about 300 to 5000 g/mol, 500 to 5000g/mol, 700 to 5000 g/mol, 500 to 2000 g/mol, 700 to 2000 g/mol, 700 to1500 g/mol, 300 to 1500 g/mol, 300 to 2000 g/mol, 400 to 1000 g/mol, 400to 900 g/mol, 400 to 800 g/mol, 500 to 900 g/mol, or 500 to 800 g/mol.In some embodiments, the weight average molecular weight of saidoligosaccharide preparation is from about 2000 to 2800 g/mol, 2100 to2700 g/mol, 2200 to 2600 g/mol, 2300 to 2500 g/mol, or 2320 to 2420g/mol. In some embodiments, the number average molecular weight of saidoligosaccharide preparation is from about 1000 to 2000 g/mol, 1100 to1900 g/mol, 1200 to 1800 g/mol, 1300 to 1700 g/mol, 1400 to 1600 g/mol,or 1450 to 1550 g/mol.

The present disclosure is based, at least in part, on the discovery thatoligosaccharides comprising one or more anhydro-subunit selectivelymodulate (e.g., promote or inhibit) the growth of microbial species andfunctions of the gut microflora. Accordingly, the disclosure features,inter alia, methods of reducing the feed conversion ratio (FCR) of ananimal by providing a feed comprising a synthetic oligosaccharide feedadditive that selectively modulates the level of microbial speciesalready present in the gut microbiome.

Provided herein are methods of promoting the growth of one or moremicrobial species in the gastrointestinal tract of an animal,comprising: administering a nutritional composition comprising a basenutritional composition and a synthetic oligosaccharide preparationdescribed herein to an animal, wherein the level of one or moremicrobial species in the gastrointestinal tract of the animal is higherrelative to the level of the microbial species in the gastrointestinaltract of an animal administered a nutritional composition lacking thesynthetic oligosaccharide preparation or relative to the level of themicrobial species in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the microbial species is a bacterial species. Inother embodiments, the microbial species is an archaea species. In otherembodiments, the microbial species is a virus, bacteriophage, orprotozoan species.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the gastrointestinal tract of theanimal. In some embodiments, the sample is a biopsy of agastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. Insome embodiments, the method further comprises detecting the level ofthe microbial species in the sample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation. In some embodiments, the method furthercomprises detecting the level of the 2nd, 3rd, 4th, or 5th microbialspecies in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a lower feed conversion ratio relativeto the feed conversion ratio of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to an animal. Insome embodiments the animal is livestock. In some embodiments the animalis a companion animal. In some embodiments, the animal is a fish (e.g.salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey), seafood(e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. nurserypig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, orhuman. In some embodiments, the animal is poultry. In some embodiments,the animal is a chicken (e.g. broiler, layer, breeder), turkey, duck, orgoose. In some embodiments, the animal is a pig (e.g. nursery pig,grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Provided herein are methods of enhancing expression of a microbialprotein within the gastrointestinal tract of an animal, comprising:administering a nutritional composition comprising a base nutritionalcomposition and a synthetic oligosaccharide preparation described hereinto the animal, wherein the expression one or more microbial protein ishigher relative to the expression level in the gastrointestinal tract ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the expression level in thegastrointestinal tract of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation, and wherein the microbial protein is a hydrolytic enzyme, aprotein involved in digestion (e.g. hydrolytic enzymatic digestion), ora protein involved in metabolism.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the animal. In some embodiments, thesample is a biopsy of a gastrointestinal tissue (e.g., a cecal biopsy)or a fecal sample. In some embodiments, the method further comprisesdetecting the expression level of the microbial protein in the sample.

In some embodiments, the level of at least 2, 3, 4, or 5 microbialproteins are higher relative to the expression level in thegastrointestinal tract of an animal administered a nutritionalcomposition lacking the synthetic oligosaccharide preparation, andwherein each of the microbial proteins is a hydrolytic enzyme, a proteininvolved in digestion (e.g. hydrolytic enzymatic digestion), or aprotein involved in metabolism. In some embodiments, the level of atleast 2, 3, 4, or 5 microbial proteins are higher relative to theexpression level in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation, and wherein each of the microbial proteinsis a hydrolytic enzyme, a protein involved in digestion (e.g. hydrolyticenzymatic digestion), or a protein involved in metabolism. In someembodiments, the method further comprises determining the level of theat least the 2nd, 3rd, 4th, or 5th microbial protein in the sample.

In some embodiments, the microbial protein is a carbohydrate activeenzyme (CAZymes). In some embodiments, the microbial protein is asus-like polysaccharide utilization loci (PULs) protein. In someembodiments, the microbial protein is selected from the group consistingof: GH127 (CAZyme α-L-arabinofuranosidase), susC (outer membrane proteininvolved in starch binding) and susD (starch binding protein). In someembodiments, the expression level of the microbial protein is determinedby analyzing RNA or protein expression.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is higher relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of one ormore microbial species in the gastrointestinal tract of the animal ishigher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare higher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the method further comprises detectingthe level of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a lower feed conversion ratio relativeto the feed conversion ratio of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments the animal is livestock. In some embodiments theanimal is a companion animal. In some embodiments, the animal is a fish(e.g. salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey),seafood (e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g.nursery pig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig,donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird,or human. In some embodiments, the animal is poultry. In someembodiments, the animal is a chicken (e.g. broiler, layer, breeder),turkey, duck, or goose. In some embodiments, the animal is a pig (e.g.nursery pig, grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Provided herein are methods of reducing the feed conversion ratio of ananimal, comprising: administering a nutritional composition comprising abase nutritional composition and a synthetic oligosaccharide preparationdescribed herein to the animal, wherein the feed conversion ratio of theanimal is lower relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the feed conversion ratio ofthe animal prior to administration of the nutritional compositioncomprising the oligosaccharide preparation.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a lower feed conversion ratio relativeto the feed conversion ratio of the animal prior to administration ofnutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is higher relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the feed conversion ratio of the animal is at least75%, 50%, 25%, 15%, 10%, 5%, or 1% less than the feed conversion ratioof an animal administered a nutritional composition lacking thesynthetic oligosaccharide. In some embodiments, the feed conversionratio of the animal is at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% lessthan the feed conversion ratio of the animal prior to administration ofthe nutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the feed efficiency of the animal is at least 75%,50%, 25%, 15%, 10%, 5%, or 1% greater than the feed efficiency of ananimal administered the nutritional composition lacking the syntheticoligosaccharide. In some embodiments, the feed efficiency of the animalis at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% greater than the feedefficiency of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the gastrointestinal tract of theanimal. In some embodiments, the sample is a biopsy of agastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. Insome embodiments, the method further comprises detecting the level ofthe microbial species in the sample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation. In some embodiments, the method furthercomprises detecting the level of the 2nd, 3rd, 4th, or 5th microbialspecies in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments the animal livestock. In some embodiments the animalis a companion animal. In some embodiments, the animal is a fish (e.g.salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey), seafood(e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. nurserypig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, orhuman. In some embodiments, the animal is poultry. In some embodiments,the animal is a chicken (e.g. broiler, layer, breeder), turkey, duck, orgoose. In some embodiments, the animal is a pig (e.g. nursery pig,grower/finisher pig). In some embodiments, the nutritional compositionis an animal feed composition. In some embodiments, the base nutritionalcomposition is base animal feed.

Provided herein are methods of increasing the feed efficiency of ananimal, comprising: administering a nutritional composition comprising abase nutritional composition and a synthetic oligosaccharide preparationdescribed herein to the animal, wherein the feed efficiency of theanimal is higher relative to the feed efficiency of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the feed efficiency of theanimal prior to administration of the nutritional composition comprisingthe synthetic oligosaccharide preparation.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is higher relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation.

In some embodiments, the feed conversion ratio of the animal is at least75%, 50%, 25%, 15%, 10%, 5%, or 1% less than the feed conversion ratioof an animal administered the nutritional composition lacking thesynthetic oligosaccharide. In some embodiments, the feed conversionratio of the animal is at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% lessthan the feed conversion ratio of the animal prior to administration ofthe nutritional composition comprising the synthetic oligosaccharide.

In some embodiments, the feed efficiency of the animal is at least 75%,50%, 25%, 15%, 10%, 5%, or 1% greater than the feed efficiency of ananimal administered the nutritional composition lacking the syntheticoligosaccharide. In some embodiments, the feed efficiency of the animalis at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% greater than the feedefficiency of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the gastrointestinal tract of theanimal. In some embodiments, the sample is a biopsy of agastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. Insome embodiments, the method further comprises detecting the level ofthe microbial species in the sample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising theoligosaccharide preparation. In some embodiments, the method furthercomprises detecting the level of the 2nd, 3rd, 4th, or 5th microbialspecies in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments the animal livestock. In some embodiments the animalis a companion animal. In some embodiments, the animal is a fish (e.g.salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey), seafood(e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. nurserypig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, orhuman. In some embodiments, the animal is poultry. In some embodiments,the animal is a chicken (e.g. broiler, layer, breeder), turkey, duck, orgoose. In some embodiments, the animal is a pig (e.g. nursery pig,grower/finisher pig). In some embodiments, the nutritional compositionis an animal feed composition. In some embodiments, the base nutritionalcomposition is base animal feed.

Provided herein are methods of increasing the body weight of an animal,comprising: administering a nutritional composition comprising a basenutritional composition and a synthetic oligosaccharide preparationdescribed herein to the animal, wherein the body weight of the animal ishigher relative to the body weight of an animal administered anutritional composition lacking the synthetic oligosaccharidepreparation or relative to the body weight of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the body weight of the animal is at least 75%, 50%,25%, 15%, 10%, 5%, or 1% greater than the body weight of the animaladministered the nutritional composition lacking the syntheticoligosaccharide. In some embodiments, the body weight of the animal isat least 75%, 50%, 25%, 15%, 10%, 5%, or 1% greater than the body weightof the animal prior to administration of nutritional compositioncomprising the synthetic oligosaccharide.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the animal has a lower feed conversionratio relative to the feed conversion ratio of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is higher relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of one ormore microbial species in the gastrointestinal tract of the animal ishigher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the feed conversion ratio of the animal is at least75%, 50%, 25%, 15%, 10%, 5%, or 1% less than the feed conversion ratioof an animal administered the nutritional composition lacking thesynthetic oligosaccharide. In some embodiments, the feed conversionratio of the animal is at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% lessthan the feed conversion ratio of the animal prior to administration ofthe nutritional composition comprising the synthetic oligosaccharide.

In some embodiments, the feed efficiency of the animal is at least 75%,50%, 25%, 15%, 10%, 5%, or 1% greater than the feed efficiency of ananimal administered the nutritional composition lacking the syntheticoligosaccharide. In some embodiments, the feed efficiency of the animalis at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% greater than the feedefficiency of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the gastrointestinal tract of theanimal. In some embodiments, the sample is a biopsy of agastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. Insome embodiments, the method further comprises detecting the level ofthe microbial species in the sample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare higher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the method further comprises detectingthe level of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments the animal livestock. In some embodiments the animalis a companion animal. In some embodiments, the animal is a fish (e.g.salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey), seafood(e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g. nurserypig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig, donkey,camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, orhuman. In some embodiments, the animal is poultry. In some embodiments,the animal is a chicken (e.g. broiler, layer, breeder), turkey, duck, orgoose. In some embodiments, the animal is a pig (e.g. nursery pig,grower/finisher pig). In some embodiments, the nutritional compositionis an animal feed composition. In some embodiments, the base nutritionalcomposition is base animal feed.

Provided herein are methods of improving animal health, comprising:administering a nutritional composition comprising a base nutritionalcomposition and a synthetic oligosaccharide preparation described hereinto the animal, and wherein the administering results in at least one ofa) improving the gastrointestinal microbiota of the animal, b)selectively increasing the level of one or more beneficial bacteria inthe gastrointestinal microbiota of the animal, c) improving at least oneof weight gain and feed conversion, and d) promoting growth of theanimal.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio relative to the feed conversion ratio of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the animal has a lower feed conversionratio relative to the feed conversion ratio of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is higher relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of one ormore microbial species in the gastrointestinal tract of the animal ishigher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation.

In some embodiments, the feed conversion ratio of the animal is at least75%, 50%, 25%, 15%, 10%, 5%, or 1% less than the feed conversion ratioof an animal administered the nutritional composition lacking thesynthetic oligosaccharide. In some embodiments, the feed conversionratio of the animal is at least 75%, 50%, 25%, 15%, 10%, 5%, or 1% lessthan the feed conversion ratio of the animal prior to administration ofthe nutritional composition comprising the synthetic oligosaccharide.

In some embodiments, the feed efficiency of the animal is at least 75%,50%, 25%, 15%, 10%, 5%, or 1% greater than the feed efficiency of ananimal administered the nutritional composition lacking the syntheticoligosaccharide.

In some embodiments, the feed efficiency of the animal is at least 75%,50%, 25%, 15%, 10%, 5%, or 1% greater than the feed efficiency of theanimal prior to administration of the nutritional composition comprisingthe synthetic oligosaccharide.

In some embodiments, the method further comprises obtaining a sample ofthe gastrointestinal microbiota of the gastrointestinal tract of theanimal. In some embodiments, the sample is a biopsy of agastrointestinal tissue (e.g., a cecal biopsy) or a fecal sample. Insome embodiments, the method further comprises detecting the level ofthe microbial species in the sample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are higher relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare higher relative to the level of the microbial species in thegastrointestinal tract of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the method further comprises detectingthe level of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is beneficial to the animal(e.g., beneficial to the health of the animal). In some embodiments, themicrobial species belongs to the genus Bacteroides, Odoribacter,Oscillibacter, Subdoligranulum, Biophila, or Barnesiella. In someembodiments, the microbial species is selected from the group consistingof: Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus,and Barnesiella intestinihominis. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is a beneficial to the animal (e.g.,beneficial to the health of the animal). In some embodiments, at leastone of the 2nd, 3rd, 4th, or 5th microbial species belongs to the genusBacteroides, Odoribacter, Oscillibacter, Subdoligranulum, Biophila, orBarnesiella. In some embodiments, at least one of the 2nd, 3rd, 4th, or5th microbial species is selected from the group consisting of:Bacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, andBarnesiella intestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one microbial species that belongs to the genus Bacteroides,Odoribacter, Oscillibacter, Subdoligranulum, Biophila, or Barnesiella.In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% ofat least one of Bacteroides clarus, Bacteroides dorei, Odoribactersplanchinicus, and Barnesiella intestinihominis.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days. In some embodiments,the nutritional composition comprising the synthetic oligosaccharidepreparation is administered to the animal at least once, twice, three,four, or five times a day. In some embodiments, administering comprisesproviding the nutritional composition to the animal to ingest at will,wherein the animal ingests at least a portion of the nutritionalcomposition in every 24-hour period.

In some embodiments, the level of the microbial species is determined byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is determined by shotgun DNA sequencing.

In some embodiments the animal is livestock. In some embodiments theanimal is a companion animal. In some embodiments, the animal is a fish(e.g. salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey),seafood (e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g.nursery pig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig,donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird,or human. In some embodiments, the animal is poultry. In someembodiments, the animal is a chicken (e.g. broiler, layer, breeder),turkey, duck, or goose. In some embodiments, the animal is a pig (e.g.nursery pig, grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Provided herein are methods of inhibiting the growth of a microbialspecies in the gastrointestinal tract of an animal, comprising:administering a nutritional composition comprising a base nutritionalcomposition and a synthetic oligosaccharide preparation described hereinto the animal, and wherein the level of one or more microbial species inthe gastrointestinal tract of the animal is lower relative to the levelof the microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the microbial species is a bacterial species. Inother embodiments, the microbial species is an archaea species. In otherembodiments, the microbial species is a virus, bacteriophage, orprotozoan species.

In some embodiments, the method comprises obtaining a sample of thegastrointestinal microbiota of the gastrointestinal tract of the animal.In some embodiments, the sample is a biopsy of a gastrointestinal tissue(e.g., a cecal biopsy) or a fecal sample. In some embodiments, themethod comprises determining the level of the microbial species in thesample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are lower relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare lower relative to the level of the microbial species in thegastrointestinal tract of an animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the methods comprise detecting thelevel of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is detrimental to the animal.In some embodiments, the microbial species is pathogenic to the animal.In some embodiments, the microbial species is pathogenic to humans butnot to animals. In some embodiment, the microbial species isHelicobacter pullorum. In some embodiments, the microbial species ispathogenic to humans but not to animals and the microbial species isHelicobacter pullorum. In some embodiments, the microbial speciesbelongs to the phylum Proteobacteria. In some embodiments, the microbialspecies belongs to the genus Helicobacter, Escherichia, Salmonella,Vibrio, or Yersinia. In some embodiments, the microbial species isselected from the group consisting of: Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Camplobacter jejuni, andLactobacillus crispatus.

In some embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis a detrimental to the animal. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is pathogenic to the animal. In someof the embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis pathogenic to humans but not to animals. In some of the embodiments,the microbial species is Helicobacter pullorum. In some embodiments, themicrobial species is pathogenic to humans but not to animals and themicrobial species is Helicobacter pullorum. In some embodiments, atleast one of the 2nd, 3rd, 4th, or 5th microbial species belongs to thephylum Proteobacteria. In some embodiments, at least one of the 2nd,3rd, 4th, or 5th microbial species belongs to the genus Helicobacter,Escherichia, Salmonella, Vibrio, or Yersinia.

In some embodiments, the animal has a gastrointestinal microbiotacomprising less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%or 0.1% Helicobacter pullorum, Proteobacteria johnsonii, Escherichiacoli, Camplobacter jejuni, and Lactobacillus crispatus.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal atleast once, twice, three, four, or five times a day. In someembodiments, administering comprises providing the nutritionalcomposition to the animal to ingest at will, wherein the animal ingestsat least a portion of the nutritional composition in every 24-hourperiod.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio (FCR) relative to the feed conversion ratio of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the animal has a lower feed conversionratio (FCR) relative to the feed conversion ratio of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the level of the microbial species is detected byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is detected by shotgun DNA sequencing.

In some embodiments, the animal is livestock. In some embodiments theanimal is a companion animal. In some embodiments, the animal is a fish(e.g. salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey),seafood (e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g.nursery pig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig,donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird,or human. In some embodiments, the animal is poultry. In someembodiments, the animal is a chicken (e.g. broiler, layer, breeder),turkey, duck, or goose. In some embodiments, the animal is a pig (e.g.nursery pig, grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Provided herein are methods of increasing survival of an animal,comprising: administering a nutritional composition comprising a basenutritional composition and a synthetic oligosaccharide preparationdescribed herein to the animal.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the microbial species is a bacterial species. Inother embodiments, the microbial species is an archaea species. In otherembodiments, the microbial species is a virus, bacteriophage, orprotozoan species.

In some embodiments, the animal is exposed to a pathogenic microbialspecies.

In some embodiments, the animal administered the nutritional compositioncomprising the synthetic oligosaccharide preparation lives longerrelative to an animal administered a nutritional composition lacking thesynthetic oligosaccharide preparation.

In some embodiments, the level of one or more microbial species in thegastrointestinal tract of the animal is lower relative to the level ofthe microbial species in the gastrointestinal tract of an animaladministered a nutritional composition lacking the syntheticoligosaccharide preparation or relative to the level of the microbialspecies in the gastrointestinal tract of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the method comprises obtaining a sample of thegastrointestinal microbiota of the gastrointestinal tract of the animal.In some embodiments, the sample is a biopsy of a gastrointestinal tissue(e.g., a cecal biopsy) or a fecal sample. In some embodiments, themethod comprises determining the level of the microbial species in thesample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are lower relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare lower relative to the level of the microbial species in thegastrointestinal tract of an animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the methods comprise detecting thelevel of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is detrimental to the animal.In some embodiments, the microbial species is pathogenic to the animal.In some embodiments, the microbial species is pathogenic to humans butnot to animals. In some embodiment, the microbial species isHelicobacter pullorum. In some embodiments, the microbial species ispathogenic to humans but not to animals and the microbial species isHelicobacter pullorum. In some embodiments, the microbial speciesbelongs to the phylum Proteobacteria. In some embodiments, the microbialspecies belongs to the genus Helicobacter, Escherichia, Salmonella,Vibrio, or Yersinia. In some embodiments, the microbial species isselected from the group consisting of: Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Camplobacter jejuni, andLactobacillus crispatus.

In some embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis a detrimental to the animal. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is pathogenic to the animals. In someof the embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis pathogenic to humans but not to animals. In some of the embodiments,the microbial species is Helicobacter pullorum. In some embodiments, themicrobial species is pathogenic to humans but not to animals and themicrobial species is Helicobacter pullorum. In some embodiments, atleast one of the 2nd, 3rd, 4th, or 5th microbial species belongs to thephylum Proteobacteria. In some embodiments, at least one of the 2nd,3rd, 4th, or 5th microbial species belongs to the genus Helicobacter,Escherichia, Salmonella, Vibrio, or Yersinia.

In some embodiments, the animal has a gastrointestinal microbiotacomprising less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%or 0.1% Helicobacter pullorum, Proteobacteria johnsonii, Escherichiacoli, Camplobacter jejuni, and Lactobacillus crispatus.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal atleast once, twice, three, four, or five times a day. In someembodiments, administering comprises providing the nutritionalcomposition to the animal to ingest at will, wherein the animal ingestsat least a portion of the nutritional composition in every 24-hourperiod.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio (FCR) relative to the feed conversion ratio of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the animal has a lower feed conversionratio (FCR) relative to the feed conversion ratio of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the level of the microbial species is detected byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is detected by shotgun DNA sequencing.

In some embodiments, the animal is livestock. In some embodiments, theanimal is a companion animal. In some embodiments, the animal is a fish(e.g. salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey),seafood (e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g.nursery pig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig,donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird,or human. In some embodiments, the animal is poultry. In someembodiments, the animal is a chicken (e.g. broiler, layer, breeder),turkey, duck, or goose. In some embodiments, the animal is a pig (e.g.nursery pig, grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Provided herein are methods of improving animal health, comprising:administering a nutritional composition comprising a base nutritionalcomposition and a synthetic oligosaccharide preparation described hereinto the animal, wherein the administering results in at least one of a)improving the gastrointestinal microbiota of the animal, b) selectivelydecreasing the level of one or more detrimental or pathogenic bacteriain the gastrointestinal microbiota of the animal, c) improving at leastone of weight gain and feed conversion ratio, and d) promoting growth ofthe animal.

In some embodiments, the synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 2; wherein each fraction comprises from 1% to90% anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry.

In some embodiments, the microbial species is a bacterial species. Inother embodiments, the microbial species is an archaea species. In otherembodiments, the microbial species is a virus, bacteriophage, orprotozoan species.

In some embodiments, the method comprises obtaining a sample of thegastrointestinal microbiota of the gastrointestinal tract of the animal.In some embodiments, the sample is a biopsy of a gastrointestinal tissue(e.g., a cecal biopsy) or a fecal sample. In some embodiments, themethod comprises determining the level of the microbial species in thesample.

In some embodiments, the level of 2, 3, 4, 5, or more microbial speciesin the gastrointestinal tract of the animal are lower relative to thelevel of the microbial species in the gastrointestinal tract of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the level of 2, 3, 4,5, or more microbial species in the gastrointestinal tract of the animalare lower relative to the level of the microbial species in thegastrointestinal tract of an animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the methods comprise detecting thelevel of the 2nd, 3rd, 4th, or 5th microbial species in the sample.

In some embodiments, the microbial species is detrimental to the animal.In some embodiments, the microbial species is pathogenic to the animal.In some embodiments, the microbial species is pathogenic to humans butnot to animals. In some embodiment, the microbial species isHelicobacter pullorum. In some embodiments, the microbial species ispathogenic to humans but not to animals and the microbial species isHelicobacter pullorum. In some embodiments, the microbial speciesbelongs to the phylum Proteobacteria. In some embodiments, the microbialspecies belongs to the genus Helicobacter, Escherichia, Salmonella,Vibrio, or Yersinia. In some embodiments, the microbial species isselected from the group consisting of: Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Camplobacter jejuni, andLactobacillus crispatus.

In some embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis a detrimental to the animal. In some embodiments, each of the 2nd,3rd, 4th, or 5th microbial species is pathogenic to the animals. In someof the embodiments, each of the 2nd, 3rd, 4th, or 5th microbial speciesis pathogenic to humans but not to animals. In some of the embodiments,the microbial species is Helicobacter pullorum. In some embodiments, themicrobial species is pathogenic to humans but not to animals and themicrobial species is Helicobacter pullorum. In some embodiments, atleast one of the 2nd, 3rd, 4th, or 5th microbial species belongs to thephylum Proteobacteria. In some embodiments, at least one of the 2nd,3rd, 4th, or 5th microbial species belongs to the genus Helicobacter,Escherichia, Salmonella, Vibrio, or Yersinia.

In some embodiments, the animal has a gastrointestinal microbiotacomprising less than 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%or 0.1% Helicobacter pullorum, Proteobacteria johnsonii, Escherichiacoli, Camplobacter jejuni, and Lactobacillus crispatus.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal forat least 1, 7, 10, 14, 30, 45, 60, 90, or 120 days.

In some embodiments, the nutritional composition comprising thesynthetic oligosaccharide preparation is administered to the animal atleast once, twice, three, four, or five times a day. In someembodiments, administering comprises providing the nutritionalcomposition to the animal to ingest at will, wherein the animal ingestsat least a portion of the nutritional composition in every 24-hourperiod.

In some embodiments, the animal has a higher body weight relative to thebody weight of an animal administered a nutritional composition lackingthe synthetic oligosaccharide preparation. In some embodiments, theanimal has a higher feed efficiency relative to the feed efficiency ofan animal administered a nutritional composition lacking the syntheticoligosaccharide preparation. In some embodiments, the animal has a lowerfeed conversion ratio (FCR) relative to the feed conversion ratio of ananimal administered a nutritional composition lacking the syntheticoligosaccharide preparation.

In some embodiments, the animal has a higher body weight relative to thebody weight of the animal prior to administration of the nutritionalcomposition comprising the synthetic oligosaccharide preparation. Insome embodiments, the animal has a higher feed efficiency relative tothe feed efficiency of the animal prior to administration of thenutritional composition comprising the synthetic oligosaccharidepreparation. In some embodiments, the animal has a lower feed conversionratio (FCR) relative to the feed conversion ratio of the animal prior toadministration of the nutritional composition comprising the syntheticoligosaccharide preparation.

In some embodiments, the level of the microbial species is detected byDNA sequencing or RNA sequencing. In some embodiments, the level of themicrobial species is detected by shotgun DNA sequencing.

In some embodiments, the animal is livestock. In some embodiments, theanimal is a companion animal. In some embodiments, the animal is a fish(e.g. salmon, tilapia, tropical fish), poultry (e.g. chicken, turkey),seafood (e.g. shrimp), sheep, cow, cattle, buffalo, bison, pig (e.g.nursery pig, grower/finisher pig), cat, dog, rabbit, goat, guinea pig,donkey, camel, horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird,or human. In some embodiments, the animal is poultry. In someembodiments, the animal is a chicken (e.g. broiler, layer, breeder),turkey, duck, or goose. In some embodiments, the animal is a pig (e.g.nursery pig, grower/finisher pig).

In some embodiments, the nutritional composition is an animal feedcomposition described herein. In some embodiments, the base nutritionalcomposition is base animal feed described herein.

Additional aspects and advantages of the present disclosure will becomereadily apparent to those skilled in this art from the followingdetailed description, wherein only illustrative embodiments of thepresent disclosure are shown and described. As will be realized, thepresent disclosure is capable of other and different embodiments, andits several details are capable of modifications in various obviousrespects, all without departing from the disclosure. Accordingly, thedrawings and description are to be regarded as illustrative in nature,and not as restrictive.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.To the extent publications and patents or patent applicationsincorporated by reference contradict the disclosure contained in thespecification, the specification is intended to supersede and/or takeprecedence over any such contradictory material.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawing (also “figure” and “FIG.” herein), of which:

FIG. 1 illustrates part of a ¹H, ¹³C-HSQC NMR spectrum ofoligosaccharide preparation 9.2.

FIG. 2 illustrates a MALDI-MS spectrum of an oligosaccharide preparationfrom Example 9.7 that demonstrates the presence of anhydro-subunits.

FIG. 3 illustrates a 1D ¹H NMR spectrum of the anhydro-DP1 component ofan oligosaccharide preparation of Example 9.

FIG. 4 illustrates a 1D APT ¹³C-NMR spectrum of an anhydro-DP1 componentof an oligosaccharide preparation of Example 9.

FIG. 5 illustrates the GC-MS chromatogram (TIC and XIC (m/z 229) plots)showing the DP2 and anhydro-DP2 components of oligosaccharidepreparation 2.9 following derivatization.

FIG. 6 illustrates a ¹H, ¹³C-HSQC spectrum of an oligosaccharidepreparation with a caramelization anhydro-subunit.

FIG. 7 illustrates a part of a MALDI-MS spectrum comparing anoligosaccharide preparation of Example 9 at different laser energies.

FIG. 8A illustrates LC-MS/MS detection of the anhydro DP2 species atconcentration of 1-80 μg/mL of an oligosaccharide preparation in water.FIG. 8B shows a linear calibration curve resulting from the LC-MS/MSdetection of FIG. 8A.

FIG. 9 illustrates the quantification of the anhydro-DP2 content ofvarious control and treated diet compositions.

FIG. 10 illustrates a 2D-¹H JRES NMR spectrum of an anhydro-subunitcontaining gluco-oligosaccharides sample.

FIG. 11 is a representative ¹H, ¹³C-HSQC NMR spectrum of ananhydro-subunit containing gluco-oligosaccharides sample with relevantresonances and assignments used for linkage distribution.

FIG. 12 illustrates an overlay of ¹H DOSY spectra of threeanhydro-subunit containing oligosaccharides.

FIG. 13 illustrates a comparison of 1,6-Anhydro-β-D-glucose (DP1-18),1,6-Anhydro-β-D-celobiose (DP2-18), and an anhydro-subunit containingoligosaccharides sample.

FIG. 14 illustrates mass chromatograms of anhydro-subunit containingoligosaccharides (top) and digested anhydro-subunit containingoligosaccharides (bottom) at selected MRMs.

FIG. 15 shows a graph of the relative abundance versus degree ofpolymerization (DP) of an oligosaccharide of Example 9. The graph showsthe oligosaccharide preparation has monotonically decreasing DPdistribution.

FIG. 16 shows a graph of the relative abundance versus degree ofpolymerization of an oligosaccharide of Example 9. The graph shows theoligosaccharide preparation has non-monotonically decreasing DPdistribution.

FIG. 17 shows microbial species (taxa) in the cecal microbiotaassociated with increased growth performance as measured by body weight.

FIG. 18 shows microbial species (taxa) in the cecal microbiotaassociated with decreased growth performance as measured by body weight.

FIG. 19 is a graph showing the ex vivo increase in the transcription ofhydrolytic digestive enzymes by microbiota provided with anoligosaccharide preparation of Example 9.

FIG. 20 is a graph showing the effect of an oligosaccharide preparationof Example 9 on the functional metagenomics of piglets.

FIG. 21 shows the reduction of undesirable members of the phylumProteobacteria in the intestinal flora of commercial broiler chickens inthe Treated Group (birds fed the treated diets containing theoligosaccharide preparation) to compared to the Control Group (birds fedthe control diets lacking the oligosaccharide preparation).

FIG. 22 is a graph showing the correlation between the relativeabundance of Escherichia coli (left) and other Escherichia species(right) and mortality in commercial broiler chickens.

FIG. 23 illustrates two DP1 and one DP2 anhydro-subunit containingoligosaccharides.

FIG. 24 illustrates an anhydro-subunit containing oligosaccharide(cellotriosan).

FIG. 25A illustrate a MALDI-MS spectrum of an oligosaccharidepreparation from Example 2 that demonstrates the presence ofanhydro-subunits. FIG. 25B illustrates a MALDI-MS spectrum of anoligosaccharide preparation from Example 2 that demonstrates thepresence of anhydro-subunits.

FIG. 26A illustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1,and DP2 species of an oligosaccharide preparation of Example 1. FIG. 26Billustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 1. FIG. 26Cillustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 1.

FIG. 27A illustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1,and DP2 species of an oligosaccharide preparation of Example 3. FIG. 27Billustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 3. FIG. 27Cillustrates LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 3.

FIG. 28A illustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1,and DP2 species of an oligosaccharide preparation of Example 4. FIG. 28Billustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 4. FIG. 28Cillustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 4.

FIG. 29A illustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1,and DP2 species of an oligosaccharide preparation of Example 7. FIG. 29Billustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 7. FIG. 29Cillustrate LC-MS/MS detection of the anhydro DP2, anhydro DP1, and DP2species of an oligosaccharide preparation of Example 7.

FIG. 30A illustrates GC-MS spectrum detection of the DP1, anhydro DP1,DP2 and anhydro DP2 fractions of an oligosaccharide preparation ofExample 1. FIG. 30B illustrates an enlargement of the DP2 and anhydro DP2 fractions as shown in FIG. 30A.

FIG. 31A illustrates GC-MS spectrum detection of the DP1, anhydro DP1,DP2 and anhydro DP2 fractions of an oligosaccharide preparation ofExample 3. FIG. 31B illustrates an enlargement of the DP2 and anhydro DP2 fractions as shown in FIG. 31A.

FIG. 32A illustrates GC-MS spectrum detection of the DP1, anhydro DP1,DP2 and anhydro DP2 fractions of an oligosaccharide preparation ofExample 4. FIG. 32B illustrates an enlargement of the DP2 and anhydro DP2 fractions as shown in FIG. 32A.

FIG. 33A illustrates GC-MS spectrum detection of the DP1, anhydro DP1,DP2 and anhydro DP2 fractions of an oligosaccharide preparation ofExample 7. FIG. 33B illustrates an enlargement of the DP2 and anhydro DP2 fractions as shown in FIG. 33A.

FIG. 34 illustrates the effect of reaction temperature, water content,and reaction time on the content of DP2 anhydro-subunit containingoligosaccharides in the oligosaccharide preparations, as compared to anoligosaccharide preparation according to Example 2.

FIG. 35 illustrates the NMR assignments of1,6-anhydro-beta-D-glucofuranose and 1,6-anhydro-beta-D-glucopyranose.

FIG. 36 illustrates MALDI-MS spectra comparing the oligosaccharidepreparation from Example 9 at different laser energies.

DETAILED DESCRIPTION

The following description and examples illustrate embodiments of thepresent disclosure in detail. It is to be understood that this presentdisclosure is not limited to the particular embodiments described hereinand as such can vary. Those of skill in the art will recognize thatthere are numerous embodiments and modifications of this presentdisclosure, which are encompassed within its scope.

All terms are intended to be understood as they would be understood by aperson skilled in the art. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood by one of ordinary skill in the art to which the disclosurepertains.

The section headings used herein are for organizational purposes onlyand are not to be construed as limiting the subject matter described.

Although various features of the present disclosure may be described inthe context of a single embodiment, the features may also be providedseparately or in any suitable combination. Conversely, although thepresent disclosure may be described herein in the context of separateembodiments for clarity, the present disclosure may also be implementedin a single embodiment.

The following definitions supplement those in the art and are directedto the current application and are not to be imputed to any related orunrelated case, e.g., to any commonly owned patent or application.Although any methods and materials similar or equivalent to thosedescribed herein can be used in the practice for testing of the presentdisclosure, the preferred materials and methods are described herein.Accordingly, the terminology used herein is for the purpose ofdescribing particular embodiments only and is not intended to belimiting.

I. Definitions

The terminology used herein is for the purpose of describing particularcases only and is not intended to be limiting. As used herein, thesingular forms “a,” “an,” and “the” are intended to include the pluralforms as well, unless the context clearly indicates otherwise.Furthermore, to the extent that the terms “including,” “includes,”“having,” “has,” “with,” or variants thereof are used in either thedetailed description and/or the claims, such terms are intended to beinclusive in a manner similar to the term “comprising.”

It is understood that terms such as “comprises,” “comprised,”“comprising,” and the like have the meaning attributed to it in U.S.Patent law; i.e., they mean “includes,” “included,” “including,” and thelike and are intended to be inclusive or open ended and does not excludeadditional, unrecited elements or method steps; and that terms such as“consisting essentially of” and “consists essentially of” have themeaning ascribed to them in U.S. Patent law; i.e., they allow forelements not explicitly recited, but exclude elements that are found inthe prior art or that affect a basic or novel characteristic of theinvention.

The term “and/or” as used in a phrase such as “A and/or B” herein isintended to include both A and B; A or B; A (alone); and B (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following embodiments: A, B, and C;A, B, or C; A or B; A or C; B or C; A and B; A and C; B and C; A(alone); B (alone); and C (alone).

When ranges are used herein for physical properties, such as molecularweight, or chemical properties, such as chemical formulae, allcombinations and sub-combinations of ranges and specific embodimentstherein are intended to be included. The term “about” when referring toa number or a numerical range means that the number or numerical rangereferred to is an approximation within experimental variability (orwithin statistical experimental error), and thus the number or numericalrange, in some instances, will vary between 1% and 15% of the statednumber or numerical range. In some embodiments, the term “about” meanswithin 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, or0.05% of a given value or range.

As used herein the term “administering” includes providing a syntheticoligosaccharide preparation, a nutritional composition, a liquid, or ananimal feed composition described herein, to an animal such that theanimal can ingest the synthetic oligosaccharide preparation, thenutritional composition, the liquid, or the animal feed composition. Insuch embodiments, the animal ingests some portion of the syntheticoligosaccharide preparation, the nutritional composition, or the animalfeed composition. In some embodiments, the synthetic oligosaccharidepreparation, the nutritional composition, the liquid, or the animal feedcomposition is provided to said animal such that the animal may ingestthe synthetic oligosaccharide preparation, the nutritional composition,the liquid, or the animal feed composition at will. In some embodiments,the synthetic oligosaccharide preparation, the nutritional composition,the liquid, or the animal feed composition is administered to saidanimal as a prescribed diet. In some embodiments, the syntheticoligosaccharide preparation, the nutritional composition, the liquid, orthe animal feed composition is administered to said animal via manualfeeding, e.g., an oral syringe feeding, a tube feeding, etc. In someembodiments, the synthetic oligosaccharide preparation, the nutritionalcomposition, the liquid, or the animal feed composition is administeredto said animal oral, e.g., at will or manually. In some embodiments, theanimal ingests some portion of the synthetic oligosaccharidepreparation, the nutritional composition, the liquid, or the animal feedcomposition in every 24-hour period or every other 24-hour period for atleast 7 days, 14 days, 21 days, 30 days, 45 days, 60 days, 75 days, 90days or 120 days. In some embodiments, the oligosaccharide preparationmay be dissolved in water or another liquid, and the animal ingests someportion of the oligosaccharide preparation by drinking the liquid. Insome embodiments, the oligosaccharide is provided to the animal via itsdrinking water. In some embodiments, the oligosaccharide preparation,nutritional composition, liquid, or animal feed composition is consumedat will.

As used herein, the term “inclusion level” or “dose” refers to theconcentration of an oligosaccharide preparation in a nutritionalcomposition, a liquid, a diet, or an animal feed composition provided tothe animal. In some embodiments, the inclusion level is measured as themass concentration of the oligosaccharide preparation in the finalnutritional composition, liquid, diet, or animal feed. For example, theinclusion level may be measured in units of parts per million (ppm) ofthe oligosaccharide on a dry solids weight basis per the total weight ofthe final nutritional composition, liquid, diet, or animal feed. Incertain embodiments, the dry solids mass of the oligosaccharidepreparation is measured as the dry-basis mass of DP1+ species. In otherembodiments, the dry solids mass of the oligosaccharide preparation ismeasured as the dry-basis mass of DP2+ species.

As used herein, the term “specific dose” refers to the quantity of anoligosaccharide preparation consumed by an animal per unit of time andrelative to its body mass. In some embodiments, the specific dose may bemeasured in units of mg of oligosaccharide preparation (on a drysolids-basis) per kg of body weight of the animal per day (i.e.,mg/kg/day).

As used herein the term “feed conversion ratio (FCR),” refers to theratio of feed mass input (for example consumed by the animal) to theanimal output, wherein the animal output is the target animal product.For example, the animal output for dairy animals is milk, whereas theanimal output for animals raised for meat is body mass.

As used herein, “feed efficiency” refers to the ratio of the animaloutput to the feed mass input (for example consumed by the animal),wherein the animal output is the target animal product.

As used herein, the term “anhydro-subunit” refers to a product ofthermal dehydration of a monosaccharide (or monosaccharide subunit) or asugar caramelization product. For example, an “anhydro-subunit” can bean anhydro-monosaccharide such as anhydro-glucose. As another example,an “anhydro-subunit” can be linked with one or more regular oranhydro-monosaccharide subunits via glycosidic linkage.

The term “oligosaccharide” refers to a monosaccharide or a compoundcontaining two or more monosaccharide subunits linked by glycosidicbonds. As such, an oligosaccharide includes a regular monosaccharide; ananhydro-monosaccharide; or a compound containing two or moremonosaccharide subunits, wherein one or more monosaccharide subunits areoptionally, independently replaced by one or more anhydro-subunits. Anoligosaccharide can be functionalized. As used herein, the termoligosaccharide encompasses all species of the oligosaccharide, whereineach of the monosaccharide subunit in the oligosaccharide isindependently and optionally functionalized and/or replaced with itscorresponding anhydro-monosaccharide subunit.

As used herein, the term “oligosaccharide preparation” refers to apreparation that comprises at least one oligosaccharide.

As used herein, the term “gluco-oligosaccharide” refers to a glucose ora compound containing two or more glucose monosaccharide subunits linkedby glycosidic bonds. As such, a gluco-oligosaccharide includes aglucose; an anhydro-glucose; or a compound containing two or moreglucose monosaccharide subunits linked by glycosidic bonds, wherein oneor more of said glucose monosaccharide subunits are each optionally andindependently replaced with an anhydro-glucose subunit.

As used herein, the term “galacto-oligosaccharide” refers to a galactoseor a compound containing two or more galactose monosaccharide subunitslinked by glycosidic bonds. As such, a galacto-oligosaccharide includesa galactose; an anhydro-galactose or a compound containing two or moregalactose monosaccharide subunits linked by glycosidic bonds, wherein atleast one monosaccharide subunit is optionally replaced with ananhydro-galactose subunit.

As used herein, the term “gluco-galacto-oligosaccharide preparation”refers to a composition that is produced from a complete or incompletesugar condensation reaction of glucose and galactose. Accordingly, insome embodiments, a gluco-galactose-oligosaccharide preparationcomprises gluco-oligosaccharides, galacto-oligosaccharides, compoundscontaining one or more glucose monosaccharide subunits and one or moregalactose monosaccharide subunits linked by glycosidic bonds, or acombination thereof. In some embodiments, agluco-galactose-oligosaccharide preparation comprisesgluco-oligosaccharides and compounds containing one or more glucosemonosaccharide subunits and one or more galactose monosaccharidesubunits linked by glycosidic bonds. In some embodiments, agluco-galactose-oligosaccharide preparation comprisesgalacto-oligosaccharides and compounds containing one or more glucosemonosaccharide subunits and one or more galactose monosaccharidesubunits linked by glycosidic bonds. In some embodiments, agluco-galactose-oligosaccharide preparation comprises compoundscontaining one or more glucose monosaccharide subunits and one or moregalactose monosaccharide subunits linked by glycosidic bonds.

As used herein, the term “monosaccharide unit” and “monosaccharidesubunit” are used interchangeably. A “monosaccharide subunit” refers toa monosaccharide monomer in an oligosaccharide. For an oligosaccharidehaving a degree of polymerization of 1, the oligosaccharide can bereferred to as a monosaccharide subunit or monosaccharide. For anoligosaccharide having a degree of polymerization of 2 or higher, itsmonosaccharide subunits are linked via glycosidic bonds.

As used herein, the term “regular monosaccharide” refers to amonosaccharide that does not contain an anhydro-subunit. The term“regular disaccharide” refers to a disaccharide that does not contain ananhydro-subunit. Accordingly, the term “regular subunit” refers to asubunit that is not an anhydro-subunit.

As used herein, the term an “anhydro DPn oligosaccharide,” an “anhydroDPn species,” or a “DPn anhydro-subunit containing oligosaccharide”refers to an oligosaccharide that has a degree of polymerization of nand comprises one or more anhydro-subunits. As such, an anhydro-glucoseis a DP1 anhydro-subunit containing oligosaccharide and a cellotriosanis a DP3 anhydro-subunit containing oligosaccharide.

The term “relative abundance” or “abundance,” as used herein, refers tothe abundance of a species in terms of how common or rare the speciesexists. For example, a DP1 fraction comprising 10% anhydro-subunitcontaining oligosaccharides by relative abundance can refer to aplurality of DP1 oligosaccharides, wherein 10% of the DP1oligosaccharides are anhydro-monosaccharides. The relative abundance,e.g., for a certain DP fraction of oligosaccharides, can be determinedby suitable analytical instrumentations, for example, mass spectrometryand liquid chromatography such as LC-MS/MS, GC-MS, HPLC-MS, andMALDI-MS. In some embodiments, the relative abundance is determined byintegrating the area under the peaks of the chromatographs (e.g.,LC-MS/MS, GC-MS, and HPLC-MS) that correspond to the fractions ofinterest. In some embodiments, the relative abundance is determined bythe peak intensities (e.g., MALDI-MS). In some embodiments, the relativeabundance is determined by a combination of analytical methods such as aweight determination after separation by liquid chromatography.

As used herein, the singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “an agent” includes a plurality of such agents,and reference to “the oligosaccharide” includes reference to one or moreoligosaccharides (or to a plurality of oligosaccharides) and equivalentsthereof known to those skilled in the art, and so forth.

II. Oligosaccharide Preparation

Disclosed herein are oligosaccharide preparations suitable for use inanimal nutritional compositions. In some embodiments, the disclosedoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than or equal to 2. In some embodiments, n is an integer greaterthan 2. In some embodiments, each of the 1 to n fractions in theoligosaccharide preparation comprises from 1% to 90% anhydro-subunitcontaining oligosaccharides by relative abundance as measured by massspectrometry. In some embodiments, the relative abundance ofoligosaccharides in each fraction decreases monotonically with itsdegree of polymerization.

In some embodiments, n is an integer greater than or equal to 3. In someembodiments, n is an integer within a range of 1 to 100, such as 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, each of the 1 ton fraction in the oligosaccharide preparation independently comprisesfrom 0.1% to 90% anhydro-subunit containing oligosaccharides by relativeabundance as measured by mass spectrometry or by LC-MS/MS or GC-MS. Insome embodiments, each of the 1 to n fraction in the oligosaccharidepreparation independently comprises from about 0.1% to about 15%anhydro-subunit containing oligosaccharides. In some embodiments, eachof the 1 to n fraction in the oligosaccharide preparation independentlycomprises from about 0.5% to about 15% anhydro-subunit containingoligosaccharides. In some embodiments, the DP1 and DP2 fractions eachindependently comprises from about 0.1% to about 15% of anhydro-subunitcontaining oligosaccharides by relative abundance as measured by massspectrometry such as MALDI-MS or by LC-MS/MS or GC-MS. In someembodiments, the DP1 and DP2 fractions each independently comprises fromabout 0.5% to about 15% of anhydro-subunit containing oligosaccharides.In some embodiments, the DP1 and DP2 fractions each independentlycomprises from about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 2% or 3% toabout 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% of anhydro-subunitcontaining oligosaccharides by relative abundance as measured by massspectrometry, LC-MS/MS or GC-MS. In some embodiments, the relativeabundance of oligosaccharides in each fraction decreases monotonicallywith its degree of polymerization.

In some embodiments, the oligosaccharide preparation is a syntheticoligosaccharide preparation. In some embodiments, a syntheticoligosaccharide preparation refers to a plurality of oligosaccharidesproduced by a process that does not require live organisms. In someembodiments, a synthetic oligosaccharide preparation refers to aplurality of oligosaccharides produced by a process that does notrequire enzymes. In some embodiments, a synthetic oligosaccharidepreparation refers to a plurality of oligosaccharides produced by achemical process. In certain embodiments, a synthetic oligosaccharidepreparation refers to a plurality of oligosaccharides produced by thecondensation of sugars.

A. Prebiotic Utility of Oligosaccharides

Disclosed herein are oligosaccharide preparations comprisinganhydro-sugar components and/or sugar dehydration product componentsthat exhibit complex functional modulation of a microbial community,such as the animal gut microbiome. The oligosaccharide preparationsprovide a utility to regulate the utilization of fermentable carbon bymicroflora and direct metabolic flux to beneficial species, thusproviding a microbiome-mediated health or nutritional benefit.

Indigestible carbohydrates can act as prebiotics by providing afermentable carbon source to a microbial community. For example, dietsrich in soluble plant fiber have been identified for their ability tonourish the gut microflora. Additionally, bifidogenic prebiotics supportthe growth of bifidobacteria (e.g., members of genus Bifidobacterium)and lactogenic prebiotics support the growth of Lactobacillus species.

Prebiotic fiber may be fermented to beneficial chemical species such asshort chain fatty acids (SCFAs). Prebiotic fibers include: resistantstarches; cellulose; pectins such as rhamnogalactans, arabinogalactans,arabinans; hemicelluloses such as arabinoxylans, xyloglucans,glucomannans, galactomannans; xylans such as corn cob oligosaccharides;b-glucans such as cereal b-glucans, yeast b-glucans, bacterialb-glucans; polyfructans such as inulin and levan; and gums such asalginate. Inulin is a common bifidogenic prebiotic fiber.

In other cases, prebiotics act by hindering the ability of pathogenicbacteria to engraft and thus infect a host organism via anti-adherencemechanisms such as the competitive binding of cell surface receptorcites. Certain galacto-oligosaccharides provide effective anti-adherenceof various enteropathogenic organisms, such as Escherichia species.

Prebiotics are typically provided to a host animal by incorporation intothe diet, upon which they exhibit a dose-dependent response (at least upto a saturation threshold). For example, providing a higher dose of abifidogenic prebiotic such as inulin tends to provide a larger increasein the population of Bifidobacterium species. Higher doses of inulincorrespond to higher production of SCFAs through fermentation. This isbecause the prebiotic provides a metabolic carbon source and more carbontranslates to more fermented product. Similarly, providing a higher doseof an anti-adherence prebiotic provides a likelihood of competitivelybinding surface receptor sites.

Certain carbohydrate species comprising modified monomeric subunits mayaffect the manner in which microbial systems utilize other carbohydratesotherwise available to them as a prebiotic source. For example, suchcarbohydrate species may be a modified carbohydrate species thatmodulate the microbial starch utilization system (SUS), i.e., proteinsresponsible for the cell-surface recognition, glycosidic cleavage, andimportation of starch metabolites.

Carbohydrate compositions capable of complex modulation of themicrobiota of animals have utility as feed additives that improve animalhealth and nutrition via their impact on the animal microbiome. Forexample, modulation of butyrate production by the gut microflora confershealth benefits to the animal by promoting a healthy gut mucosa, barrierfunction, and via anti-inflammatory effects. Modulation of propionicacid production affects the metabolic energy extracted from the animal'sdiet via increased gluconeogenesis. Relevant microbial communitiesinclude, for example, ileal, jejunal, and cecal and/or fecal microbiotain poultry, pigs, dogs, cats, horses, or the ruminant microbiota ofcattle, cows, sheep, etc. Other microbial communities include the skinmicroflora, nasal microflora, etc.

Further, disclosed herein are oligosaccharide preparations that areadvantageous in that they can be selectively analyzed and quantified ina complex nutritional composition such as complete animal feed due tothe presence of anhydro-subunits. It is of commercial utility to assayfor the presence and/or concentration of feed additives such asoligosaccharide preparations. Such assay may be performed for thepurpose of quality control, to determine whether the additive wasblended consistently with the base nutritional composition to provide afinal nutritional composition comprising the additive at the intendeddose or level of inclusion.

However, the nutritional compositions themselves comprise a largequantity and diversity of carbohydrate structures (e.g., starch, plantfibers and pectins). It is therefore particularly challenging todistinguish small quantities of oligosaccharide-based feed additivesfrom the vast sea of other carbohydrates present as base of thenutritional composition. As such, the herein disclosed oligosaccharidepreparation provides a means to distinguish itself from othercarbohydrates sources in the nutritional composition through theanhydro-subunits.

B. Degree of Polymerization (DP) Distribution

In some embodiments, the oligosaccharide preparation comprises at leastn fractions of oligosaccharides each having a distinct degree ofpolymerization selected from 1 to n (DP1 to DPn fractions). In someembodiments, the oligosaccharide preparation comprises n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions). For example, in someembodiments, the DP1 fraction comprises one or more monosaccharidesand/or one or more anhydro-monosaccharides. As another example, in someembodiments, the DP1 fraction comprises glucose, galactose, fructose,1,6-anhydro-β-D-glucofuranose, 1,6-anhydro-β-D-glucopyranose, or anycombination thereof. As yet another example, in some embodiments, theDP2 fraction comprises one or more regular disaccharides and one or moreanhydro-subunit containing disaccharides. In some embodiments, the DP2fraction comprises lactose.

In some embodiments, n is at least 2, at least 3, at least 5, at least6, at least 7, at least 8, at least 9, at least 10, at least 11, atleast 12, at least 13, at least 14, at least 15, at least 16, at least17, at least 18, at least 19, at least 20, at least 21, at least 22, atleast 23, at least 24, at least 25, at least 26, at least 27, at least28, at least 29, at least 30, at least 31, at least 32, at least 33, atleast 34, at least 35, at least 36, at least 37, at least 38, at least39, at least 40, at least 41, at least 42, at least 43, at least 44, atleast 45, at least 46, at least 47, at least 48, at least 49, at least50, at least 51, at least 52, at least 53, at least 54, at least 55, atleast 56, at least 57, at least 58, at least 59, at least 60, at least61, at least 62, at least 63, at least 64, at least 65, at least 66, atleast 67, at least 68, at least 69, at least 70, at least 71, at least72, at least 73, at least 74, at least 75, at least 76, at least 77, atleast 78, at least 79, at least 80, at least 81, at least 82, at least83, at least 84, at least 85, at least 86, at least 87, at least 88, atleast 89, at least 90, at least 91, at least 92, at least 93, at least94, at least 95, at least 96, at least 97, at least 98, at least 99, orat least 100. In some embodiments, n is 2, 3, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100. Insome embodiments, n is less than 10, less than 11, less than 12, lessthan 13, less than 14, less than 15, less than 16, less than 17, lessthan 18, less than 19, less than 20, less than 21, less than 22, lessthan 23, less than 24, less than 25, less than 26, less than 27, lessthan 28, less than 29, less than 30, less than 31, less than 32, lessthan 33, less than 34, less than 35, less than 36, less than 37, lessthan 38, less than 39, less than 40, less than 41, less than 42, lessthan 43, less than 44, less than 45, less than 46, less than 47, lessthan 48, less than 49, less than 50, less than 51, less than 52, lessthan 53, less than 54, less than 55, less than 56, less than 57, lessthan 58, less than 59, less than 60, less than 61, less than 62, lessthan 63, less than 64, less than 65, less than 66, less than 67, lessthan 68, less than 69, less than 70, less than 71, less than 72, lessthan 73, less than 74, less than 75, less than 76, less than 77, lessthan 78, less than 79, less than 80, less than 81, less than 82, lessthan 83, less than 84, less than 85, less than 86, less than 87, lessthan 88, less than 89, less than 90, less than 91, less than 92, lessthan 93, less than 94, less than 95, less than 96, less than 97, lessthan 98, less than 99, or less than 100. In some embodiments, n is from2 to 100, from 5 to 90, from 10 to 90, from 10 to 80, from 10 to 70,from 10 to 60, from 10 to 50, from 10 to 40, from 10 to 30, from 15 to60, from 15 to 50, from 15 to 45, from 15 to 40, from 15 to 35, or from15 to 30.

A distribution of the degree of polymerization of the oligosaccharidepreparation can be determined by any suitable analytical method andinstrumentation, including but not limited to end group method, osmoticpressure (osmometry), ultracentrifugation, viscosity measurements, lightscattering method, size exclusion chromatography (SEC), SEC-MALLS, fieldflow fractionation (FFF), asymmetric flow field flow fractionation(A4F), high-performance liquid chromatography (HPLC), and massspectrometry (MS). For example, the distribution of the degree ofpolymerization may be determined and/or detected by mass spectrometry,such as matrix-assisted laser desorption/ionization (MALDI)-MS, liquidchromatography (LC)-MS, or gas chromatography (GC)-MS. For anotherexample, the distribution of the degree of polymerization can bedetermined and/or detected by SEC, such as gel permeation chromatography(GPC). As yet another example, the distribution of the degree ofpolymerization can be determined and/or detected by HPLC, FFF, or A4F.In some embodiments, the distribution of the degree of polymerization isdetermined and/or detected by MALDI-MS. In some embodiments, thedistribution of the degree of polymerization is determined and/ordetected by GC-MS or LC-MS. In some embodiments, the distribution of thedegree of polymerization is determined and/or detected by SEC. In someembodiments, the distribution of the degree of polymerization isdetermined and/or detected by HPLC. In some embodiments, thedistribution of the degree of polymerization is determined and/ordetected by a combination of analytical instrumentations such asMALDI-MS and SEC. In some embodiments, the degree of polymerization ofthe oligosaccharide preparation can be determined based on its molecularweight and molecular weight distribution. For example, FIG. 2 shows aMALDI-MS spectrum that illustrates the degrees of polymerizations ofvarious fractions and the presence of anhydro-subunit containingoligosaccharides (the −18 g/mol MW offset peaks) in all of the observedfractions.

In some embodiments, the relative abundance of oligosaccharides in amajority of the fractions decreases monotonically with its degree ofpolymerization. In some embodiments, the relative abundance ofoligosaccharides of less than 6, less than 5, less than 4, less than 3,or less than 2 fractions of the oligosaccharide preparation do notdecrease monotonically with its degree of polymerization.

In some embodiments, the relative abundance of oligosaccharides in atleast 5, at least 10, at least 15, at least 20, at least 25, at least30, at least 35, at least 40, at least 45, or at least 50 DP fractionsdecreases monotonically with its degree of polymerization. In someembodiments, the relative abundance of oligosaccharides in at least 5,at least 10, at least 15, at least 20, at least 25, at least 30, atleast 35, at least 40, at least 45, or at least 50 consecutive DPfractions decreases monotonically with its degree of polymerization. Insome embodiments, the relative abundance of oligosaccharides in at least5, at least 10, at least 20, or at least 30 DP fractions decreasesmonotonically with its degree of polymerization. In some embodiments,the relative abundance of oligosaccharides in at least 5, at least 10,at least 20, or at least 30 consecutive DP fractions decreasesmonotonically with its degree of polymerization.

In some embodiments, the relative abundance of oligosaccharides in eachof the n fractions decreases monotonically with its degree ofpolymerization. For example, FIG. 15 provides an example of a DPdistribution where the relative abundance of oligosaccharides in each ofthe n fractions decrease monotonically with its DP. For example, in someembodiments, only the relative abundance of oligosaccharides in the DP3fraction does not decrease monotonically with its degree ofpolymerization, i.e., the relative abundance of oligosaccharides in theDP3 fraction is lower than the relative abundance of oligosaccharides inthe DP4 fraction. In some embodiments, the relative abundance ofoligosaccharides in the DP2 fraction is lower than the relativeabundance of oligosaccharides in the DP3 fraction. For example, FIG. 16illustrates a degree of polymerization distribution wherein the relativeabundance of oligosaccharides in the DP2 fraction does not decreasemonotonically with its degree of polymerization.

In some embodiments, a herein described oligosaccharide preparation hasa DP1 fraction content of from about 1% to about 50%, from about 1% toabout 40%, from about 1% to about 35%, from about 1% to about 30%, fromabout 1% to about 25%, from about 1% to about 20%, from about 1% toabout 15%, from about 5% to about 50%, from about 5% to about 40%, fromabout 5% to about 35%, from about 5% to about 30%, from about 5% toabout 25%, from about 5% to about 20%, from about 5% to about 15%, fromabout 10% to about 50%, from about 10% to about 40%, from about 10% toabout 35%, from about 10% to about 30%, from about 10% to about 25%,from about 10% to about 20%, or from about 10% to about 15% by weight orby relative abundance. In some embodiments, the oligosaccharidepreparation has a DP1 fraction content of from about 10% to about 35%,from about 10% to about 20%, or from about 10% to about 15% by weight orby relative abundance. In some embodiments, the content of the DP1fraction is determined by MALDI-MS. In some embodiments, the content ofthe DP1 fraction is determined by HPLC. In some embodiments, the contentof the DP1 fraction is determined by LC-MS/MS or GC-MS.

In some embodiments, a herein described oligosaccharide preparation hasa DP2 fraction content of from about 1% to about 35%, from about 1% toabout 30%, from about 1% to about 25%, from about 1% to about 20%, fromabout 1% to about 15%, from about 1% to about 10%, from about 5% toabout 30%, from about 5% to about 25%, from about 5% to about 20%, fromabout 5% to about 15%, or from about 5% to about 10% by weight or byrelative abundance. In some embodiments, the oligosaccharide preparationhas a DP2 fraction content of from about 5% to about 25%, from about 5%to about 20%, from about 5% to about 15%, or from about 5% to about 10%by weight or by relative abundance. In some embodiments, the content ofthe DP2 fraction is determined by MALDI-MS. In some embodiments, thecontent of the DP2 fraction is determined by HPLC. In some embodiments,the content of the DP2 fraction is determined by LC-MS/MS or GC-MS.

In some embodiments, a herein described oligosaccharide preparation hasa DP3 fraction content of from about 1% to about 30%, from about 1% toabout 25%, from about 1% to about 20%, from about 1% to about 15%, fromabout 1% to about 10%, from about 5% to about 30%, from about 5% toabout 25%, from about 5% to about 20%, from about 5% to about 15%, orfrom about 5% to about 10% by weight or by relative abundance. In someembodiments, the oligosaccharide preparation has a DP3 fraction contentof from about 1% to about 15%, from about 1% to about 10%, from about 5%to about 15%, or from about 5% to about 10% by weight or by relativeabundance. In some embodiments, the content of the DP3 fraction isdetermined by MALDI-MS. In some embodiments, the content of the DP3fraction is determined by HPLC. In some embodiments, the content of theDP3 fraction is determined by LC-MS/MS or GC-MS.

In some embodiments, a herein described oligosaccharide preparation hasa DP4 fraction content of from about 0.1% to about 20%, from about 0.1%to about 15%, from about 0.1% to about 10%, from about 0.1% to about 5%,from about 1% to about 20%, from about 1% to about 15%, from about 1% toabout 10%, or from about 1% to about 5% by weight or by relativeabundance. In some embodiments, the oligosaccharide preparation has aDP4 fraction content of from about 1% to about 15%, from about 1% toabout 10%, or from about 1% to about 5% by weight or by relativeabundance. In some embodiments, a herein described oligosaccharidepreparation has a DP5 fraction content of from about 0.1% to about 15%,from about 0.1% to about 10%, from about 0.1% to about 5%, from about 1%to about 15%, from about 1% to about 10%, or from about 1% to about 5%by weight or by relative abundance. In some embodiments, theoligosaccharide preparation has a DP5 fraction content of from about 1%to about 10% or from about 1% to about 5% by weight or by relativeabundance. In some embodiments, the content of the DP4 and/or the DP5fraction is determined by MALDI-MS. In some embodiments, the content ofthe DP4 and/or the DP5 fraction is determined by HPLC. In someembodiments, the content of the DP4 and/or the DP5 fraction isdetermined by LC-MS/MS or GC-MS.

In some embodiments, the ratio of DP2 fraction to DP1 fraction in theoligosaccharide preparation is from about 0.01 to about 0.8, from about0.02 to about 0.7, from about 0.02 to about 0.6, from about 0.02 toabout 0.5, from about 0.02 to about 0.4, from about 0.02 to about 0.3,from about 0.02 to about 0.2, from about 0.1 to about 0.6, from about0.1 to about 0.5, from about 0.1 to about 0.4, or from about 0.1 toabout 0.3 by their weight or relative abundance. In some embodiments,the ratio of DP2 fraction to DP1 fraction in the oligosaccharidepreparation is from about 0.02 to about 0.4 by their weight or relativeabundance.

In some embodiments, the ratio of DP3 fraction to DP2 fraction in theoligosaccharide preparation is from about 0.01 to about 0.7, from about0.01 to about 0.6, from about 0.01 to about 0.5, from about 0.01 toabout 0.4, from about 0.01 to about 0.3, or from about 0.01 to about 0.2by their weight or relative abundance. In some embodiments, the ratio ofDP3 fraction to DP2 fraction in the oligosaccharide preparation is fromabout 0.01 to about 0.3 by their weight or relative abundance.

In some embodiments, the aggregate content of DP1 and DP2 fractions inthe oligosaccharide preparation is less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, or less than 10%by weight or by relative abundance. In some embodiments, the aggregatecontent of DP1 and DP2 fractions in the oligosaccharide preparation isless than 50%, less than 30%, or less than 10% by weight or by relativeabundance.

In some embodiments, an oligosaccharide preparation described herein hasa mean DP value within a range of 2 to 10. In some embodiments, theoligosaccharide preparation has a mean DP value of from about 2 to about8, from about 2 to about 5, or from about 2 to about 4. In someembodiments, the oligosaccharide preparation has a mean DP value ofabout 3.5. The mean DP value can be determined by SEC or by elementalanalysis.

C. Anhydro-Subunit Level

In some embodiments, each of the n fractions of oligosaccharidesindependently comprises an anhydro-subunit level. For instance, in someembodiments, the DP1 fraction comprises 10% anhydro-subunit containingoligosaccharides by relative abundance, and the DP2 fraction comprises15% anhydro-subunit containing oligosaccharides by relative abundance.For another example, in some embodiments, DP1, DP2, and DP3 fractioneach comprises 5%, 10%, and 2% anhydro-subunit containingoligosaccharides by relative abundance, respectively. In otherembodiments, two or more fractions of oligosaccharides may comprisesimilar level of anhydro-subunit containing oligosaccharides. Forexample, in some embodiments, the DP1 and DP3 fraction each comprisesabout 5% anhydro-subunit containing oligosaccharides by relativeabundance.

In some embodiments, each of the 1 to n fractions in a herein describedoligosaccharide preparation independently comprises from about 0.1% to15% of anhydro-subunit containing oligosaccharides by relative abundanceas measured by mass spectrometry, LC-MS/MS or GC-MS. In someembodiments, each of the 1 to n fractions in the oligosaccharidepreparation independently comprises from about 0.5% to 15% ofanhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry, LC-MS/MS or GC-MS. In some embodiments,LC-MS/MS is used to determine the relative abundance foroligosaccharides in the DP1, DP2, and/or DP3 fractions. In someembodiments, GC-MS is used to determine the relative abundance foroligosaccharides in the DP1, DP2, and/or DP3 fractions. In someembodiments, MALDI-MS is used to determine the relative abundance foroligosaccharides in the DP4 fraction or in a higher DP fraction. In someembodiments, the relative abundance of a certain fraction is determinedby integrating the area under the peaks of the LC-MS/MS chromatogramthat are designated as corresponding to that fraction. In someembodiments, the relative abundance of a certain fraction is determinedby integrating the area under the peaks of the GC-MS chromatogram thatare designated as corresponding to that fraction.

The level of anhydro-subunits can be determined by any suitableanalytical methods, such as nuclear magnetic resonance (NMR)spectroscopy, mass spectrometry, HPLC, FFF, A4F, or any combinationthereof. In some embodiments, the level of anhydro-subunits isdetermined, at least in part, by mass spectrometry such as MALDI-MS. Insome embodiments, the level of anhydro-subunits is determined, at leastin part, by NMR. In some embodiments, the level of anhydro-subunitscontaining oligosaccharides is determined, at least in part, by HPLC. Insome embodiments, the level of anhydro-subunits containingoligosaccharides is determined by MALDI-MS, as illustrated by the −18g/mol MW offset peaks in FIG. 2. In some embodiments, the presence andthe type of species of anhydro-subunits can be determined and/ordetected by NMR, as illustrated by Example 11, FIG. 3, and FIG. 4. Insome embodiments, the relative abundance of anhydro-subunit containingoligosaccharides is determined by MALDI-MS. In some embodiments, therelative abundance of anhydro-subunit containing oligosaccharides isdetermined by LC-MS/MS, as illustrated in FIGS. 26A-26C, 27A-27C,28A-28C and 29A-29C. In some embodiments, the relative abundance ofanhydro-subunit containing oligosaccharides is determined by GC-MS, asillustrated in FIGS. 30A-30B, 31A-31B, 32A-32B and 33A-33B.

In some embodiments, at least one fraction of a herein describedoligosaccharide preparation comprises less than 80%, less than 70%, lessthan 60%, less than 50%, less than 40%, less than 30%, less than 20%,less than 19%, less than 18%, less than 17%, less than 16%, less than15%, less than 14%, less than 13%, less than 12%, less than 11%, lessthan 10%, less than 9%, less than 8%, less than 7%, less than 6%, lessthan 5%, less than 4%, less than 3%, less than 2%, or less than 1% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, at least one fraction of a herein describedoligosaccharide preparation comprises less than 10%, less than 9%, lessthan 8%, less than 7%, less than 6%, less than 5%, less than 4%, lessthan 3%, or less than 2% of anhydro-subunit containing oligosaccharidesby relative abundance. In other embodiments, at least one fraction of aherein described oligosaccharide preparation comprises greater than0.5%, greater than 0.8%, greater than 1%, greater than 2%, greater than3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 11%,greater than 12%, greater than 13%, greater than 14%, greater than 15%,greater than 16%, greater than 17%, greater than 18%, greater than 19%,greater than 20%, greater than 30%, greater than 40%, greater than 50%,greater than 60%, greater than 70%, or greater than 80% ofanhydro-subunit containing oligosaccharides by relative abundance. Inother embodiments, at least one fraction of a herein describedoligosaccharide preparation comprises greater than 20%, greater than21%, greater than 22%, greater than 23%, greater than 24%, greater than25%, greater than 26%, greater than 27%, greater than 28%, greater than29%, or greater than 30% of anhydro-subunit containing oligosaccharidesby relative abundance. In some embodiments, at least one fraction (suchas DP1, DP2, and/or DP3) of the oligosaccharide preparation comprisesabout 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%,about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%,about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about24%, about 25%, or about 30% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, at leastone fraction (such as DP1, DP2, and/or DP3) of the oligosaccharidepreparation comprises about 0.1%, about 0.2%, about 0.3%, about 0.4%,about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%,about 1.5%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, or about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, at leastone fraction (such as DP1, DP2, and/or DP3) of the oligosaccharidepreparation comprises from about 0.1% to about 90%, from about 0.5% toabout 90%, from about 0.5% to about 80%, from about 0.5% to about 70%,from about 0.5% to about 60%, from about 0.5% to about 50%, from about0.5% to about 40%, from about 0.5% to about 30%, from about 0.5% toabout 20%, from about 0.5% to about 10%, from about 0.5% to about 9%,from about 0.5% to about 8%, from about 0.5% to about 7%, from about0.5% to about 6%, from about 0.5% to about 5%, from about 0.5% to about4%, from about 0.5% to about 3%, from about 0.5% to about 2%, from about1% to about 10%, from about 2% to about 9%, from about 2% to about 8%,from about 2% to about 7%, from about 2% to about 6%, from about 2% toabout 5%, from about 2% to about 4%, from about 2% to about 3%, or fromabout 5% to about 10% of anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, the DP1 and DP2 fractions ofthe oligosaccharide preparation each independently comprisesanhydro-subunit containing oligosaccharides within a range of from about0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%to about 8%, 9%, 10%, 11%, 12%, or 15% by relative abundance as measuredby mass spectrometry, LC-MS/MS, or GC-MS. In some embodiments, the DP1and DP2 fractions each independently comprises from about 0.5% to about15% of anhydro-subunit containing oligosaccharides by relative abundanceas measured by mass spectrometry or by LC-MS/MS or GC-MS.

In some embodiments, each fraction of a herein described oligosaccharidepreparation comprises less than 80%, less than 70%, less than 60%, lessthan 50%, less than 40%, less than 30%, less than 20%, less than 19%,less than 18%, less than 17%, less than 16%, less than 15%, less than14%, less than 13%, less than 12%, less than 11%, less than 10%, lessthan 9%, less than 8%, less than 7%, less than 6%, less than 5%, lessthan 4%, less than 3%, or less than 2% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, eachfraction of a herein described oligosaccharide preparation comprisesless than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2% anhydro-subunitcontaining oligosaccharides by relative abundance. In other embodiments,each fraction of a herein described oligosaccharide preparationcomprises greater than 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 30%, 40%, 50%, 60%, 70%, or 80%of anhydro-subunit containing oligosaccharides by relative abundance. Inother embodiments, each fraction of a herein described oligosaccharidepreparation comprises greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, or 30% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, each fraction of a hereindescribed oligosaccharide preparation comprises about 0.1%, about 0.2%,about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%,about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%,about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, orabout 30% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, each fraction of a herein describedoligosaccharide preparation comprises about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, or about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, eachfraction of a herein described oligosaccharide preparation comprisesfrom about 0.1% to about 90%, from about 0.1% to about 15%, from about0.5% to about 90%, from about 0.5% to about 80%, from about 0.5% toabout 70%, from about 0.5% to about 60%, from about 0.5% to about 50%,from about 0.5% to about 40%, from about 0.5% to about 30%, from about0.5% to about 20%, from about 0.5% to about 10%, from about 0.5% toabout 9%, from about 0.5% to about 8%, from about 0.5% to about 7%, fromabout 0.5% to about 6%, from about 0.5% to about 5%, from about 0.5% toabout 4%, from about 0.5% to about 3%, from about 0.5% to about 2%, fromabout 2% to about 9%, from about 2% to about 8%, from about 2% to about7%, from about 2% to about 6%, from about 2% to about 5%, from about 2%to about 4%, from about 2% to about 3%, or from about 5% to about 10% ofanhydro-subunit containing oligosaccharides by relative abundance.

In some embodiments, a herein described oligosaccharide preparationcomprises less than 80%, less than 70%, less than 60%, less than 50%,less than 40%, less than 30%, less than 20%, less than 19%, less than18%, less than 17%, less than 16%, less than 15%, less than 14%, lessthan 13%, less than 12%, less than 11%, less than 10%, less than 9%,less than 8%, less than 7%, less than 6%, less than 5%, less than 4%,less than 3%, less than 2%, or less than 1% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,the oligosaccharide preparation comprises less than 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, or 2% anhydro-subunit containing oligosaccharides byrelative abundance. In other embodiments, the oligosaccharidepreparation comprises greater than 0.5%, greater than 0.8%, greater than1%, greater than 2%, greater than 3%, greater than 4%, greater than 5%,greater than 6%, greater than 7%, greater than 8%, greater than 9%,greater than 10%, greater than 11%, greater than 12%, greater than 13%,greater than 14%, greater than 15%, greater than 16%, greater than 17%,greater than 18%, greater than 19%, greater than 20%, greater than 30%,greater than 40%, greater than 50%, greater than 60%, greater than 70%,or greater than 80% anhydro-subunit containing oligosaccharides byrelative abundance. In other embodiments, the oligosaccharidepreparation comprises greater than 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, or 30% anhydro-subunit containing oligosaccharides byrelative abundance. In some embodiments, the oligosaccharide preparationcomprises about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%,about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%,about 23%, about 24%, about 25%, or about 30% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,the oligosaccharide preparation comprises about 0.1%, about 0.2%, about0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about7%, about 8%, about 9%, or about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, theoligosaccharide preparation comprises from about 0.1% to about 90%, fromabout 0.1% to about 15%, from about 0.5% to about 90%, from about 0.5%to about 80%, from about 0.5% to about 70%, from about 0.5% to about60%, from about 0.5% to about 50%, from about 0.5% to about 40%, fromabout 0.5% to about 30%, from about 0.5% to about 20%, from about 0.5%to about 10%, from about 0.5% to about 9%, from about 0.5% to about 8%,from about 0.5% to about 7%, from about 0.5% to about 6%, from about0.5% to about 5%, from about 0.5% to about 4%, from about 0.5% to about3%, from about 0.5% to about 2%, from about 2% to about 9%, from about2% to about 8%, from about 2% to about 7%, from about 2% to about 6%,from about 2% to about 5%, from about 2% to about 4%, from about 2% toabout 3%, or from about 5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance.

In some embodiments, the DP1 fraction of a herein describedoligosaccharide preparation comprises less than 30%, less than 20%, lessthan 19%, less than 18%, less than 17%, less than 16%, less than 15%,less than 14%, less than 13%, less than 12%, less than 11%, less than10%, less than 9%, less than 8%, less than 7%, less than 6%, less than5%, less than 4%, less than 3%, less than 2%, or less than 1% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, the DP1 fraction of a herein described oligosaccharidepreparation comprises greater than 0.1%, greater than 0.5%, greater than0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 11%,greater than 12%, greater than 13%, greater than 14%, or greater than15% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, the DP1 fraction of a herein describedoligosaccharide preparation comprises about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,the DP1 fraction of a herein described oligosaccharide preparationcomprises from about 0.1% to about 15%, from about 0.1% to about 20%,from about 0.5% to about 20%, from 0.5% to about 10%, from about 0.5% toabout 15%, from about 1% to about 20%, from about 1% to about 15%, fromabout 1% to about 10%, from about 2% to about 14%, from about 3% toabout 13%, from about 4% to about 12%, from about 5% to about 11%, fromabout 5% to about 10%, from about 6% to about 9%, or from about 7% toabout 8% of anhydro-subunit containing oligosaccharides by relativeabundance, or any ranges therebetween. In some embodiments, the DP1fraction of a herein described oligosaccharide preparation comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, therelative abundance of anhydro-subunit containing oligosaccharides isdetermined by mass spectrometry such as MALDI-MS. In some embodiments,the relative abundance of anhydro-subunit containing oligosaccharides isdetermined by LC-MS/MS. In some embodiments, the relative abundance ofanhydro-subunit containing oligosaccharides is determined by GC-MS.

In some embodiments, the DP2 fraction of a herein describedoligosaccharide preparation comprises less than 30%, less than 20%, lessthan 19%, less than 18%, less than 17%, less than 16%, less than 15%,less than 14%, less than 13%, less than 12%, less than 11%, less than10%, less than 9%, less than 8%, less than 7%, less than 6%, less than5%, less than 4%, less than 3%, less than 2%, or less than 1% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, the DP2 fraction of a herein described oligosaccharidepreparation comprises greater than 0.1%, greater than 0.5%, greater than0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 11%,greater than 12%, greater than 13%, greater than 14%, or greater than15% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, the DP2 fraction of a herein describedoligosaccharide preparation comprises about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,the DP2 fraction of a herein described oligosaccharide preparationcomprises from about 0.1% to about 15%, from about 0.1% to about 20%,from about 0.5% to about 20%, from 0.5% to about 10%, from about 0.5% toabout 15%, from about 1% to about 20%, from about 1% to about 15%, fromabout 1% to about 10%, from about 2% to about 14%, from about 3% toabout 13%, from about 4% to about 12%, from about 5% to about 11%, fromabout 0.5% to about 10%, from about 6% to about 9%, or from about 7% toabout 8% of anhydro-subunit containing oligosaccharides by relativeabundance, or any ranges therebetween. In some embodiments, the DP2fraction of a herein described oligosaccharide preparation comprisesfrom about 5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, therelative abundance of anhydro-subunit containing oligosaccharides isdetermined by mass spectrometry such as MALDI-MS. In some embodiments,the relative abundance of anhydro-subunit containing oligosaccharides isdetermined by LC-MS/MS. In some embodiments, the relative abundance ofanhydro-subunit containing oligosaccharides is determined by GC-MS.

In some embodiments, the DP3 fraction of a herein describedoligosaccharide preparation comprises less than 30%, less than 20%, lessthan 19%, less than 18%, less than 17%, less than 16%, less than 15%,less than 14%, less than 13%, less than 12%, less than 11%, less than10%, less than 9%, less than 8%, less than 7%, less than 6%, less than5%, less than 4%, less than 3%, less than 2%, or less than 1% ofanhydro-subunit containing oligosaccharides by relative abundance. Insome embodiments, the DP3 fraction of a herein described oligosaccharidepreparation comprises greater than 0.1%, greater than 0.5%, greater than0.8%, greater than 1%, greater than 1.5%, greater than 2%, greater than3%, greater than 4%, greater than 5%, greater than 6%, greater than 7%,greater than 8%, greater than 9%, greater than 10%, greater than 11%,greater than 12%, greater than 13%, greater than 14%, or greater than15% of anhydro-subunit containing oligosaccharides by relativeabundance. In some embodiments, the DP3 fraction of a herein describedoligosaccharide preparation comprises about 0.5%, about 1%, about 2%,about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%,about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about16%, about 17%, about 18%, about 19%, or about 20% of anhydro-subunitcontaining oligosaccharides by relative abundance. In some embodiments,the DP3 fraction of a herein described oligosaccharide preparationcomprises from about 0.1% to about 15%, from about 0.1% to about 20%,from about 0.5% to about 20%, from 0.5% to about 10%, from about 0.5% toabout 15%, from about 1% to about 20%, from about 1% to about 15%, fromabout 1% to about 10%, from about 2% to about 14%, from about 3% toabout 13%, from about 4% to about 12%, from about 5% to about 11%, fromabout 5% to about 10%, from about 6% to about 9%, or from about 7% toabout 8% of anhydro-subunit containing oligosaccharides by relativeabundance, or any ranges therebetween. In some embodiments, the DP3fraction of a herein described oligosaccharide preparation comprisesfrom about 0.5% to about 10% of anhydro-subunit containingoligosaccharides by relative abundance. In some embodiments, therelative abundance of anhydro-subunit containing oligosaccharides isdetermined by mass spectrometry such as MALDI-MS. In some embodiments,the relative abundance of anhydro-subunit containing oligosaccharides isdetermined by LC-MS/MS. In some embodiments, the relative abundance ofanhydro-subunit containing oligosaccharides is determined by GC-MS.

In some embodiments, an anhydro-subunit containing oligosaccharidecomprises one or more anhydro-subunits. For instance, a DP1anhydro-subunit containing oligosaccharide comprises oneanhydro-subunit. In some embodiments, a DPn anhydro-subunit containingoligosaccharide may comprise from 1 to n anhydro-subunits. For example,in some embodiments, a DP2 anhydro-subunit containing oligosaccharidecomprises one or two anhydro-subunits. In some embodiments, eacholigosaccharide in the oligosaccharide preparation independentlycomprises zero, one, or two anhydro-subunits. In some embodiments, morethan 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%,35%, or 30% of the anhydro-subunit containing oligosaccharides have onlyone anhydro-subunit. In some embodiments, more than 99%, 95%, 90%, 85%,or 80% of the anhydro-subunit containing oligosaccharides have only oneanhydro-subunit.

In some embodiments, one or more oligosaccharides in the oligosaccharidepreparation or in each fraction of the oligosaccharide preparationcomprise 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 anhydro-subunits each linkedvia a glycosidic bond, wherein the glycosidic bonds linking eachanhydro-subunit are independently chosen. In some embodiments, one ormore oligosaccharides in the oligosaccharide preparation or in eachfraction of the oligosaccharide preparation comprise 1, 2, or 3anhydro-subunits each linked via a glycosidic bond, wherein theglycosidic bond linking each anhydro-subunit are independently chosen.In some embodiments, greater than 50%, 60%, 70%, 80%, 90%, or 99% ofoligosaccharides in the oligosaccharide preparation or in each fractioncomprise 1, 2, or 3 anhydro-subunits each linked via a glycosidic bond,wherein the glycosidic bond linking each anhydro-subunit areindependently chosen. In some embodiments, one or more oligosaccharidesin the oligosaccharide preparation or in each fraction comprise 1anhydro-subunit linked via a glycosidic bond. In some embodiments,greater than 50%, greater than 60%, greater than 70%, greater than 80%,greater than 90%, or greater than 99% of oligosaccharides in theoligosaccharide preparation or in each fraction comprise 1anhydro-subunit linked via a glycosidic bond.

D. Anhydro-Subunit Species

In some embodiments, the oligosaccharide preparation comprises differentspecies of anhydro-subunits. In some embodiments, exemplaryanhydro-subunit containing oligosaccharides are illustrated in FIG. 35,FIG. 23, and FIG. 24. In some embodiments, the oligosaccharidepreparation comprises one or more anhydro-subunits that are products ofthermal dehydration of monosaccharides, i.e., anhydro-monosaccharidesubunits. In some embodiments, the oligosaccharide preparation comprisesone or more anhydro-subunits that are products of reversible thermaldehydration of monosaccharides.

It is to be understood that an anhydro-monosaccharide (or ananhydro-monosaccharide subunit) refers to one or more species of thethermal dehydration products of the monosaccharide. For example, in someembodiments, an anhydro-glucose refers to 1,6-anhydro-β-D-glucopyranose(levoglucosan) or 1,6-anhydro-β-D-glucofuranose. In some embodiments, aplurality of anhydro-glucose refers to a plurality of1,6-anhydro-β-D-glucopyranose (levoglucosan), a plurality of1,6-anhydro-β-D-glucofuranose, a plurality of other thermal dehydrationproducts of glucose, or any combination thereof. Similarly, in someembodiments, a plurality of anhydro-galactose refers to a plurality ofany thermal dehydration products of galactose, or any combinationthereof.

In some embodiments, an oligosaccharide preparation as described hereincomprises one or more anhydro-glucose, anhydro-galactose,anhydro-mannose, anhydro-allose, anhydro-altrose, anhydro-gulose,anhydro-indose, anhydro-talose, anhydro-fructose, anhydro-ribose,anhydro-arabinose, anhydro-rhamnose, anhydro-lyxose, anhydro-xylose, orany combination of these subunits. In some embodiments, theoligosaccharide preparation comprises one or more anhydro-glucose,anhydro-galactose, anhydro-mannose, or anhydro-fructose subunits. Insome embodiments, an oligosaccharide preparation as described hereincomprises one or more of:1,6-anhydro-3-O-β-D-glucopyranosyl-β-D-glucopyranose,1,6-anhydro-3-O-α-D-glucopyranosyl-β-D-glucopyranose,1,6-anhydro-2-O-β-D-glucopyranosyl-β-D-glucopyranose,1,6-anhydro-2-O-α-D-glucopyranosyl-β-D-glucopyranose,1,6-anhydro-β-D-cellobiose (cellobiosan), 1,6-anhydro-β-D-cellotriose(cellotriosan), 1,6-anhydro-β-D-cellotetraose (cellotetraosan),1,6-anhydro-β-D-cellopentaose (cellopentaosan), and1,6-anhydro-β-D-maltose (maltosan).

In some embodiments, the oligosaccharide preparation comprises one ormore 1,6-anhydro-β-D-glucofuranose subunits. In some embodiments, theoligosaccharide preparation comprises one or more1,6-anhydro-β-D-glucopyranose (levoglucosan) subunits. For example, FIG.35 illustrates two DP1 anhydro-subunit containing oligosaccharides(levoglucosan and 1,6-anhydro-β-D-glucofuranose) and a DP2anhydro-subunit containing oligosaccharide (anhydro-cellobiose).

The presence and the level of a species of anhydro-subunit may varybased on the feed sugars used to manufacture the oligosaccharide. Forinstance, in some embodiments, gluco-oligosaccharides compriseanhydro-glucose subunits, galacto-oligosaccharides compriseanhydro-galactose subunits, and gluco-galacto-oligosaccharides compriseanhydro-glucose and anhydro-galactose subunits.

In some embodiments, the oligosaccharide preparation comprises both1,6-anhydro-β-D-glucofuranose and 1,6-anhydro-β-D-glucopyranoseanhydro-subunits. In some embodiments, at least 0.1%, 1%, 5%, 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of anhydro-subunits areselected from a group consisting of 1,6-anhydro-β-D-glucofuranose and1,6-anhydro-β-D-glucopyranose. In some embodiments, at least 1%, 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of anhydro-subunits are1,6-anhydro-β-D-glucofuranose. In some embodiments, at least 1%, 5%,10%, 20%, 30%, 40%, 50%, or 60% of anhydro-subunits are1,6-anhydro-β-D-glucopyranose.

In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is from about 10:1 to 1:10, 9:1 to 1:10,8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6,10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in thepreparation. In some embodiments, the ratio of1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in the preparation. In someembodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is about 2:1 in the preparation.

In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is about from 10:1 to 1:10, 9:1 to 1:10,8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6,10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 ineach fraction. In some embodiments, the ratio of1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in each fraction. In some embodiments,the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is about 2:1 in each fraction.

In some embodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is about from 10:1 to 1:10, 9:1 to 1:10,8:1 to 1:10, 7:1 to 1:10, 6:1 to 1:10, 5:1 to 1:10, 4:1 to 1:10, 3:1 to1:10, 2:1 to 1:10, 10:1 to 1:9, 10:1 to 1:8, 10:1 to 1:7, 10:1 to 1:6,10:1 to 1:5, 10:1 to 1:4, 10:1 to 1:3, 10:1 to 1:2, or 1:1 to 3:1 in atleast one fraction. In some embodiments, the ratio of1,6-anhydro-β-D-glucofuranose to 1,6-anhydro-β-D-glucopyranose is about10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:8, 1:9, or 1:10 in at least one fraction. In someembodiments, the ratio of 1,6-anhydro-β-D-glucofuranose to1,6-anhydro-β-D-glucopyranose is about 2:1 in at least one fraction.

In some embodiments, a herein described oligosaccharide preparationcomprises anhydro-subunit containing DP2 oligosaccharides. In someembodiments, the oligosaccharide preparation comprises anhydro-lactose,anhydro-sucrose, anhydro-cellobiose, or a combination thereof. In someembodiment, the oligosaccharide preparation comprises from about 2 to20, 2 to 15, 5 to 20, 5 to 15, or 5 to 10 species of DP2 anhydro-subunitcontaining oligosaccharides. In some embodiments, an oligosaccharidepreparation described herein does not comprise cellobiosan or does notcomprise a detectable level of cellobiosan.

In some embodiments, a herein described oligosaccharide preparationcomprises one or more anhydro-subunits that are sugar caramelizationproducts. In some embodiments, the oligosaccharide preparation comprisesone or more anhydro-subunits are sugar caramelization products selectedfrom the group consisting of: methanol; ethanol; furan; methyl glyoxal;2-methyl furan; vinyl acetate; glycolaldehyde; acetic acid; acetol;furfural; 2-furanmethanol; 3-furanmethanol; 2-hydroxycyclopent-2-en-1-one; 5-methyl furfural; 2(5H)-furanone; 2 methylcyclopentenolone; levoglucosenone; cyclic hydroxyl lactone;1,4,3,6-dianhydro-α-D-glucopyranose; dianhydro glucopyranose; and5-hydroxy methyl furfural (5-hmf). In some embodiments, theoligosaccharide preparation comprises 5-hmf anhydro-subunits.

In some embodiments, in the oligosaccharide preparation or in at leastone of the DP fractions, the anhydro-subunits that are caramelizationproducts are less abundant than the anhydro-subunits that are productsof thermal dehydration of a monosaccharide. In some embodiments, in theoligosaccharide preparation or in at least one of the fractions, theanhydro-subunits that are caramelization products are more abundant thanthe anhydro-subunits that are products of thermal dehydration of amonosaccharide. In some embodiments, in the oligosaccharide preparationor in at least one of the fractions, anhydro-subunits that arecaramelization products and anhydro-subunits that are products ofthermal dehydration of a monosaccharide have similar abundance.

In some embodiments, from about 0.01% to about 50%, from about 0.01% toabout 40%, from about 0.01% to about 30%, from about 0.01% to about 20%,from about 0.01% to about 10%, from about 0.01% to about 5%, from about0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% toabout 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%,from about 0.1% to about 50%, from about 0.1% to about 40%, from about0.1% to about 30%, from about 0.1% to about 20%, from about 0.1% toabout 10%, from about 0.1% to about 5%, from about 0.1% to about 4%,from about 0.1% to about 3%, from about 0.1% to about 2%, from about0.1% to about 1%, or from about 0.1% to about 0.5% of theanhydro-subunits in a herein described oligosaccharide preparation arecaramelization products. In some embodiments, from about 0.1% to about5%, from about 0.1% to about 2%, or from about 0.1% to about 1% of theanhydro-subunits in the oligosaccharide preparation are caramelizationproducts. In some embodiments, less than 50%, less than 40%, less than30%, less than 25%, less than 20%, less than 15%, less than 14%, lessthan 13%, less than 12%, less than 11%, less than 10%, less than 9%,less than 8%, less than 7%, less than 6%, less than 5%, less than 4%,less than 3%, less than 2%, or less than 1% of the anhydro-subunits inthe oligosaccharide preparation are caramelization products.

In some embodiments, from about 0.01% to about 50%, from about 0.01% toabout 40%, from about 0.01% to about 30%, from about 0.01% to about 20%,from about 0.01% to about 10%, from about 0.01% to about 5%, from about0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% toabout 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%,from about 0.1% to about 50%, from about 0.1% to about 40%, from about0.1% to about 30%, from about 0.1% to about 20%, from about 0.1% toabout 10%, from about 0.1% to about 5%, from about 0.1% to about 4%,from about 0.1% to about 3%, from about 0.1% to about 2%, from about0.1% to about 1%, or from about 0.1% to about 0.5% of theanhydro-subunits in at least one fraction (e.g., DP1, DP2 and/or DP3) ofa herein described preparation are caramelization products. In someembodiments, from about 0.1% to about 5%, from about 0.1% to about 2%,or from about 0.1% to about 1% of the anhydro-subunits in at least onefraction (e.g., DP1, DP2 and/or DP3) of the preparation arecaramelization products. In some embodiments, less than 50%, 40%, 30%,25%, 20%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%,or 1% of the anhydro-subunits in at least one fraction of thepreparation are caramelization products. In some embodiments, less than20%, less than 15%, less than 14%, less than 13%, less than 12%, lessthan 11%, less than 10%, less than 9%, less than 8%, less than 7%, lessthan 6%, less than 5%, less than 4%, less than 3%, less than 2%, or lessthan 1% of the anhydro-subunits in the DP1, DP2, and/or DP3 fractions ofa herein described oligosaccharide preparation are caramelizationproducts.

In some embodiments, from about 0.01% to about 50%, from about 0.01% toabout 40%, from about 0.01% to about 30%, from about 0.01% to about 20%,from about 0.01% to about 10%, from about 0.01% to about 5%, from about0.01% to about 4%, from about 0.01% to about 3%, from about 0.01% toabout 2%, from about 0.01% to about 1%, from about 0.01% to about 0.5%,from about 0.1% to about 50%, from about 0.1% to about 40%, from about0.1% to about 30%, from about 0.1% to about 20%, from about 0.1% toabout 10%, from about 0.1% to about 5%, from about 0.1% to about 4%,from about 0.1% to about 3%, from about 0.1% to about 2%, from about0.1% to about 1%, or from about 0.1% to about 0.5% of theanhydro-subunits in each fraction of a herein described oligosaccharidepreparation are caramelization products. In some embodiments, from about0.1% to about 5%, from about 0.1% to about 2%, or from about 0.1% toabout 1% of the anhydro-subunits in each fraction of the preparation arecaramelization products. In some embodiments, less than 50%, less than40%, less than 30%, less than 20%, less than 25%, less than 20%, lessthan 15%, less than 14%, less than 13%, less than 12%, less than 11%,less than 10%, less than 9%, less than 8%, less than 7%, less than 6%,less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%of the anhydro-subunits in each fraction of the preparation arecaramelization products.

In some embodiments, each of the oligosaccharides in a herein describedoligosaccharide preparation independently and optionally comprises ananhydro-subunit. In some embodiments, two or more independentoligosaccharides comprise the same or different anhydro-subunits. Insome embodiments, two or more independent oligosaccharides comprisedifferent anhydro-subunits. For example, in some embodiments, theoligosaccharide preparation comprises a DP1 anhydro-subunit containingoligosaccharide that comprises a 1,6-anhydro-β-D-glucopyranose and a DP2anhydro-subunit containing oligosaccharide that comprises a1,6-anhydro-β-D-glucofuranose subunit. In some embodiments, one or moreoligosaccharides in the oligosaccharide preparation comprise two or morethe same or different anhydro-subunits.

In some embodiments, in any fraction of the oligosaccharide preparationthat has a degree of polymerization equal or greater than 2 (i.e., DP2to DPn fractions), an anhydro-subunit may be linked to one or moreregular or anhydro-subunits. In some embodiments, in the DP2 to DPnfractions, at least one anhydro-subunit is linked to one, two, or threeother regular or anhydro-subunits. In some embodiments, in the DP2 toDPn fractions, at least one anhydro-subunit is linked to one or tworegular subunits. In some embodiments, in the DP2 to DPn fractions, atleast one anhydro-subunit is linked to one regular subunit. In someembodiments, in any of the DP2 to DPn fractions, more than 99%, 90%,80%, 70%, 60%, 50%, 40%, or 30% of anhydro-subunits are linked to oneregular subunit. In some embodiments, in each of the DP2 to DPnfraction, more than 99%, 90%, 80%, 70%, 60%, 50%, 40%, or 30% ofanhydro-subunits are linked to one regular subunit.

In some embodiments, in any fraction of the oligosaccharide preparationthat has a degree of polymerization equal or greater than 2 (i.e., DP2to DPn fractions), an anhydro-subunit can be located at a chain-end ofan oligosaccharide. In some embodiments, in any fraction of theoligosaccharide preparation that has a degree of polymerization equal orgreater than 3 (i.e., DP3 to DPn fractions), an anhydro-subunit can belocated at a position that is not a chain-end of an oligosaccharide. Insome embodiments, in the DP2 to DPn fractions, at least one of theanhydro-subunits is located at the chain-end of an oligosaccharide. Insome embodiments, greater than 99%, 95%, 90%, 85%, 80%, 75%, 70%, 65%,60%, 55%, 50%, 45%, 40%, 35%, or 30% of the anhydro-subunits in the DP2to DPn fractions are located at the chain-end of the oligosaccharides.In some embodiments, greater than 95%, 90%, 80%, 70%, 60%, 50%, 40%,30%, 20%, or 10% of the anhydro-subunits in the oligosaccharidepreparation are located at the chain-end of the oligosaccharides. Insome embodiments, greater than 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%,or 99% of the anhydro-subunit containing oligosaccharides comprise achain-end anhydro-subunit. In some embodiments, greater than 80%, 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of theanhydro-subunit containing oligosaccharides comprise a chain-endanhydro-subunit.

E. Glycosidic Linkages

In some embodiments, a herein described oligosaccharide preparationcomprises a variety of glycosidic linkages. The type and distribution ofthe glycosidic linkages can depend on the source and manufacturingmethod of the oligosaccharide preparation. In some embodiments, the typeand distribution of various glycosidic linkages can be determined and/ordetected by any suitable methods known in the art such as NMR. Forexample, in some embodiments, the glycosidic linkages are determinedand/or detected by ¹H NMR, ¹³C NMR, 2D NMR such as 2D JRES, HSQC, HMBC,DOSY, COSY, ECOSY, TOCSY, NOESY, or ROESY, or any combination thereof.In some embodiments, the glycosidic linkages are determined and/ordetected, at least in part, by ¹H NMR. In some embodiments, theglycosidic linkages are determined and/or detected, at least in part, by¹³C NMR. In some embodiments, the glycosidic linkages are determinedand/or detected, at least in part, by 2D ¹H, ¹³C-HSQC NMR.

In some embodiments, a herein described oligosaccharide preparationcomprises one or more α-(1,2) glycosidic linkages, α-(1,3) glycosidiclinkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages,β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4)glycosidic linkages, β-(1,6) glycosidic linkages, α-(1,1)-α glycosidiclinkages, α-(1,1)-β glycosidic linkages, β-(1,1)-β glycosidic linkages,or any combination thereof.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 60 mol %, from about 5%to about 55 mol %, from about 5% to about 50 mol %, from about 5% toabout 45 mol %, from about 5% to about 40 mol %, from about 5% to about35 mol %, from about 5% to about 30 mol %, from about 5% to about 25 mol%, from about 10% to about 60 mol %, from about 10% to about 55 mol %,from about 10% to about 50 mol %, from about 10% to about 45 mol %, fromabout 10% to about 40 mol %, from about 10% to about 35 mol %, fromabout 15% to about 60 mol %, from about 15% to about 55 mol %, fromabout 15% to about 50 mol %, from about 15% to about 45 mol %, fromabout 15% to about 40 mol %, from about 15% to about 35 mol %, fromabout 20% to about 60 mol %, from about 20% to about 55 mol %, fromabout 20% to about 50 mol %, from about 20% to about 45 mol %, fromabout 20% to about 40 mol %, from about 20% to about 35 mol %, fromabout 25% to about 60 mol %, from about 25% to about 55 mol %, fromabout 25% to about 50 mol %, from about 25% to about 45 mol %, fromabout 25% to about 40 mol %, or from about 25% to about 35 mol % ofα-(1,6) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 50 mol %, from about 0to about 40 mol %, from about 0 to about 35 mol %, from about 0 to about30 mol %, from about 0 to about 25 mol %, from about 0 to about 20 mol%, from about 5% to about 40 mol %, from about 5% to about 35 mol %,from about 5% to about 30 mol %, from about 5% to about 25 mol %, fromabout 5% to about 20 mol %, from about 10% to about 40 mol %, from about10% to about 35 mol %, from about 10% to about 20 mol %, from about 15%to about 40 mol %, from about 15% to about 35 mol %, from about 15% toabout 30 mol %, from about 15% to about 25 mol %, or from about 15% toabout 20 mol % of α-(1,3) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 35 mol %, from about 0 to about 30 mol %, from about 0 to about25 mol %, from about 0 to about 20 mol %, from about 0 to about 15 mol%, from about 0 to about 10 mol %, from about 2% to about 30 mol %, fromabout 2% to about 25 mol %, from about 2% to about 20 mol %, from about2% to about 15 mol %, from about 2% to about 10 mol %, from about 3% toabout 30 mol %, from about 3% to about 25 mol %, from about 3% to about20 mol %, from about 3% to about 15 mol %, from about 3% to about 10 mol%, from about 5% to about 30 mol %, from about 5% to about 25 mol %,from about 5% to about 20 mol %, from about 5% to about 15 mol %, orfrom about 5% to about 10 mol % of α-(1,2) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 30 mol %, from about 0 to about 25 mol %, from about 0 to about20 mol %, from about 0 to about 15 mol %, from about 0 to about 10 mol%, or from about 0 to about 5 mol % of α-(1,4) glycosidic linkages. Insome embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of less than 40 mol %, less than 30 mol %, lessthan 20 mol %, less than 15 mol %, less than 10 mol %, less than 9 mol%, less than 8 mol %, less than 7 mol %, less than 6 mol %, less than 5mol %, less than 4 mol %, less than 3 mol %, or less than 2 mol % ofα-(1,4) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 35 mol %, from about 0 to about 30 mol %, from about 0 to about25 mol %, from about 0 to about 20 mol %, from about 0 to about 15 mol%, from about 0 to about 10 mol %, from about 2% to about 30 mol %, fromabout 2% to about 25 mol %, from about 2% to about 20 mol %, from about2% to about 15 mol %, from about 2% to about 10 mol %, from about 5% toabout 30 mol %, from about 5% to about 25 mol %, from about 5% to about20 mol %, from about 5% to about 15 mol %, from about 5% to about 10 mol%, from about 8% to about 30 mol %, from about 8% to about 25 mol %,from about 8% to about 20 mol %, from about 8% to about 15 mol %, orfrom about 10% to about 15 mol % of β-(1,6) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 35 mol %, from about 0 to about 30 mol %, from about 0 to about25 mol %, from about 0 to about 20 mol %, from about 0 to about 15 mol%, from about 0 to about 10 mol %, from about 2% to about 30 mol %, fromabout 2% to about 25 mol %, from about 2% to about 20 mol %, from about2% to about 15 mol %, from about 2% to about 10 mol %, from about 3% toabout 30 mol %, from about 3% to about 25 mol %, from about 3% to about20 mol %, from about 3% to about 15 mol %, from about 3% to about 10 mol%, from about 5% to about 30 mol %, from about 5% to about 25 mol %,from about 5% to about 20 mol %, from about 5% to about 15 mol %, orfrom about 5% to about 10 mol % of β-(1,4) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 30 mol %, from about 0 to about 25 mol %, from about 0 to about20 mol %, from about 0 to about 15 mol %, from about 0 to about 10 mol%, from about 0 to about 5 mol %, from about 1% to about 20 mol %, fromabout 1% to about 15 mol %, from about 1% to about 10 mol %, from about1% to about 5 mol %, from about 2% to about 20 mol %, from about 2% toabout 15 mol %, from about 2% to about 10 mol %, or from about 2% toabout 5 mol % of β-(1,2) glycosidic linkages. In some embodiments, theoligosaccharide preparations have a glycosidic bond type distribution ofless than 40 mol %, less than 30 mol %, less than 20 mol %, less than 15mol %, less than 10 mol %, less than 9 mol %, less than 8 mol %, lessthan 7 mol %, less than 6 mol %, less than 5 mol %, less than 4 mol %,less than 3 mol %, or less than 2 mol % of β-(1,2) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution of from about 0 to about 40 mol %, from about 0to about 30 mol %, from about 0 to about 25 mol %, from about 0 to about20 mol %, from about 0 to about 15 mol %, from about 0 to about 10 mol%, from about 0 to about 5 mol %, from about 1% to about 20 mol %, fromabout 1% to about 15 mol %, from about 1% to about 10 mol %, from about1% to about 5 mol %, from about 2% to about 20 mol %, from about 2% toabout 15 mol %, from about 2% to about 10 mol %, or from about 2% toabout 5 mol % of β-(1,3) glycosidic linkages. In some embodiments, theoligosaccharide preparations have a glycosidic bond type distribution ofless than 40 mol %, less than 30 mol %, less than 20 mol %, less than 15mol %, less than 10 mol %, less than 9 mol %, less than 8 mol %, lessthan 7 mol %, less than 6 mol %, less than 5 mol %, less than 4 mol %,less than 3 mol %, or less than 2 mol % of β-(1,3) glycosidic linkages.

In some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution that is different from a glycosidic bond typedistribution of non-synthetic oligosaccharide preparations. For example,in some embodiments, the oligosaccharide preparations have a glycosidicbond type distribution that is different from that of the basenutritional compositions. In some embodiments, the base nutritionalcompositions comprise a natural carbohydrate source, such as starch andplant fibers. Some of the natural carbohydrate sources have a highpercentage of α-(1,4), α-(1,6), and/or β-(1,6) glycosidic linkages.Accordingly, in some embodiments, the oligosaccharide preparations havea lower percentage of α-(1,4) glycosidic linkages than the basenutritional composition. In some embodiments, the oligosaccharidepreparations have a lower percentage of α-(1,6) glycosidic linkages thanthe base nutritional composition. In other embodiments, theoligosaccharide preparations have a higher percentage of α-(1,6)glycosidic linkages than the base nutritional composition. In someembodiments, the oligosaccharide preparations have a lower percentage ofβ-(1,6) glycosidic linkages than the base nutritional composition. Insome embodiments, the oligosaccharide preparation comprises glycosidiclinkages that are not readily digestible or hydrolysable by enzymes.

Specifically, in some embodiments, the α-(1,2), α-(1,3), α-(1,4),α-(1,6), β-(1,2), β-(1,3), β-(1,4), and/or β-(1,6) glycosidic linkagesin the glycosidic bond type distribution of a herein describedoligosaccharide preparations is at least 50 mol %, at least 40 mol %, atleast 30 mol %, at least 20 mol %, at least 15 mol %, at least 10 mol %,at least 5 mol %, at least 2 mol %, or at least 1 mol % lower than thatof the base nutritional composition. In some embodiments, the α-(1,2),α-(1,3), α-(1,4), α-(1,6), β-(1,2), β-(1,3), β-(1,4), and/or β-(1,6)glycosidic linkages in the glycosidic bond type distribution of theoligosaccharide preparations is at least 50 mol %, at least 40 mol %, atleast 30 mol %, at least 20 mol %, at least 15 mol %, at least 10 mol %,at least 5 mol %, at least 2 mol %, or at least 1 mol % higher than thatof the base nutritional composition.

It should be understood by one of skill in the art that certain types ofglycosidic linkages may not be applicable to oligosaccharides comprisingcertain type of monosaccharides. For example, in some embodiments, theoligosaccharide preparation comprises α-(1,2) glycosidic linkages andα-(1,6) glycosidic linkages. In other embodiments, the oligosaccharidepreparation comprises α-(1,2) glycosidic linkages and β-(1,3) glycosidiclinkages. In some embodiments, the oligosaccharide preparation comprisesα-(1,2) glycosidic linkages, α-(1,3) glycosidic linkages, and β-(1,6)glycosidic linkages. In some embodiments, the oligosaccharidepreparation comprises α-(1,2) glycosidic linkages, α-(1,3) glycosidiclinkages, α-(1,4) glycosidic linkages, α-(1,6) glycosidic linkages,β-(1,2) glycosidic linkages, β-(1,3) glycosidic linkages, β-(1,4)glycosidic linkages, and β-(1,6) glycosidic linkages

F. Molecular Weight

The molecular weight and molecular weight distribution of theoligosaccharide preparation may be determined by any suitable analyticalmeans and instrumentation, such as end group method, osmotic pressure(osmometry), ultracentrifugation, viscosity measurements, lightscattering method, SEC, SEC-MALLS, FFF, A4F, HPLC, and massspectrometry. In some embodiments, the molecular weight and molecularweight distribution are determined by mass spectrometry, such asMALDI-MS, LC-MS, or GC-MS. In some embodiments, the molecular weight andmolecular weight distribution are determined by size exclusionchromatography (SEC), such as gel permeation chromatography (GPC). Inother embodiments, the molecular weight and molecular weightdistribution are determined by HPLC. In some embodiments, the molecularweight and molecular weight distribution are determined by MALDI-MS.

In some embodiments, a herein described oligosaccharide preparation hasa weight average molecular weight of from about 100 to about 10000g/mol, from about 200 to about 8000 g/mol, from about 300 to about 5000g/mol, from about 500 to about 5000 g/mol, from about 700 to about 5000g/mol, from about 900 to about 5000 g/mol, from about 1100 to about 5000g/mol, from about 1300 to about 5000 g/mol, from about 1500 to about5000 g/mol, from about 1700 to about 5000 g/mol, from about 300 to about4500 g/mol, from about 500 to about 4500 g/mol, from about 700 to about4500 g/mol, from about 900 to about 4500 g/mol, from about 1100 to about4500 g/mol, from about 1300 to about 4500 g/mol, from about 1500 toabout 4500 g/mol, from about 1700 to about 4500 g/mol, from about 1900to about 4500 g/mol, from about 300 to about 4000 g/mol, from about 500to about 4000 g/mol, from about 700 to about 4000 g/mol, from about 900to about 4000 g/mol, from about 1100 to about 4000 g/mol, from about1300 to about 4000 g/mol, from about 1500 to about 4000 g/mol, fromabout 1700 to about 4000 g/mol, from about 1900 to about 4000 g/mol,from about 300 to about 3000 g/mol, from about 500 to about 3000 g/mol,from about 700 to about 3000 g/mol, from about 900 to about 3000 g/mol,from about 1100 to about 3000 g/mol, from about 1300 to about 3000g/mol, from about 1500 to about 3000 g/mol, from about 1700 to about3000 g/mol, from about 1900 to about 3000 g/mol, from about 2100 toabout 3000 g/mol, from about 300 to about 2500 g/mol, from about 500 toabout 2500 g/mol, from about 700 to about 2500 g/mol, from about 900 toabout 2500 g/mol, from about 1100 to about 2500 g/mol, from about 1300to about 2500 g/mol, from about 1500 to about 2500 g/mol, from about1700 to about 2500 g/mol, from about 1900 to about 2500 g/mol, fromabout 2100 to about 2500 g/mol, from about 300 to about 1500 g/mol, fromabout 500 to about 1500 g/mol, from about 700 to about 1500 g/mol, fromabout 900 to about 1500 g/mol, from about 1100 to about 1500 g/mol, fromabout 1300 to about 1500 g/mol, from about 2000 to about 2800 g/mol,from about 2100 to about 2700 g/mol, from about 2200 to about 2600g/mol, from about 2300 to about 2500 g/mol, or from about 2320 to about2420 g/mol. In some embodiments, the weight average molecular weight ofthe oligosaccharide preparation is from about 2000 to about 2800 g/mol,from about 2100 to about 2700 g/mol, from about 2200 to about 2600g/mol, from about 2300 to about 2500 g/mol, or from about 2320 to about2420 g/mol. In some embodiments, the oligosaccharide preparation has aweight average molecular weight in a range from at least 500 g/mol, 750g/mol, 1000 g/mol, or 1500 g/mol to at most 1750 g/mol, 2000 g/mol, 2250g/mol, 2500 g/mol, or 3000 g/mol. In some embodiments, the weightaverage molecular weight of a herein described oligosaccharidepreparation is determined by HPLC according to Example 9.

In some embodiments, a herein described oligosaccharide preparation hasa number average molecular weight of from about 100 to about 10000g/mol, from about 200 to about 8000 g/mol, from about 300 to about 5000g/mol, from about 500 to about 5000 g/mol, from about 700 to about 5000g/mol, from about 900 to about 5000 g/mol, from about 1100 to about 5000g/mol, from about 1300 to about 5000 g/mol, from about 1500 to about5000 g/mol, from about 1700 to about 5000 g/mol, from about 300 to about4500 g/mol, from about 500 to about 4500 g/mol, from about 700 to about4500 g/mol, from about 900 to about 4500 g/mol, from about 1100 to about4500 g/mol, from about 1300 to about 4500 g/mol, from about 1500 toabout 4500 g/mol, from about 1700 to about 4500 g/mol, from about 1900to about 4500 g/mol, from about 300 to about 4000 g/mol, from about 500to about 4000 g/mol, from about 700 to about 4000 g/mol, from about 900to about 4000 g/mol, from about 1100 to about 4000 g/mol, from about1300 to about 4000 g/mol, from about 1500 to about 4000 g/mol, fromabout 1700 to about 4000 g/mol, from about 1900 to about 4000 g/mol,from about 300 to about 3000 g/mol, from about 500 to about 3000 g/mol,from about 700 to about 3000 g/mol, from about 900 to about 3000 g/mol,from about 1100 to about 3000 g/mol, from about 1300 to about 3000g/mol, from about 1500 to about 3000 g/mol, from about 1700 to about3000 g/mol, from about 1900 to about 3000 g/mol, from about 2100 toabout 3000 g/mol, from about 300 to about 2500 g/mol, from about 500 toabout 2500 g/mol, from about 700 to about 2500 g/mol, from about 900 toabout 2500 g/mol, from about 1100 to about 2500 g/mol, from about 1300to about 2500 g/mol, from about 1500 to about 2500 g/mol, from about1700 to about 2500 g/mol, from about 1900 to about 2500 g/mol, fromabout 2100 to about 2500 g/mol, from about 300 to about 2000 g/mol, fromabout 500 to about 300 to 2000 g/mol, from about 700 to about 2000g/mol, from about 900 to about 2000 g/mol, from about 1100 to about 2000g/mol, from about 300 to about 1500 g/mol, from about 500 to about 1500g/mol, from about 700 to about 1500 g/mol, from about 900 to about 1500g/mol, from about 1100 to about 1500 g/mol, from about 1300 to about1500 g/mol, from about 1000 to about 2000 g/mol, from about 1100 toabout 1900 g/mol, from about 1200 to about 1800 g/mol, from about 1300to about 1700 g/mol, from about 1400 to about 1600 g/mol, or from about1450 to about 1550 g/mol. In some embodiments, the number averagemolecular weight of the oligosaccharide preparation is from about 1000to about 2000 g/mol, from about 1100 to about 1900 g/mol, from about1200 to about 1800 g/mol, from about 1300 to about 1700 g/mol, 1400 to1600 g/mol, or 1450-1550 g/mol. In some embodiments, the oligosaccharidepreparation has a number average molecular weight in a range from atleast 500 g/mol, 750 g/mol, 1000 g/mol, or 1500 g/mol to at most 1750g/mol, 2000 g/mol, 2250 g/mol, 2500 g/mol, or 3000 g/mol. In someembodiments, the number average molecular weight of a herein describedoligosaccharide preparation is determined by HPLC according to Example9.

G. Types of Oligosaccharides

The species of oligosaccharides present in an oligosaccharidepreparation can depend on the type of the one or more feed sugars. Forexample, in some embodiments, the oligosaccharide preparations comprisea gluco-oligosaccharide when the feed sugars comprise glucose. Forexample, in some embodiments, the oligosaccharide preparations comprisea galacto-oligosaccharide when the feed sugars comprise galactose. Foranother example, in some embodiments, the oligosaccharide preparationscomprise gluco-galacto-oligosaccharides when the feed sugars comprisegalactose and glucose.

In some embodiments, a herein described oligosaccharide preparationcomprises one or more species of monosaccharide subunits. In someembodiments, the oligosaccharide preparation comprises oligosaccharideswith 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, or more different species of monosaccharides subunits.

In some embodiments, the oligosaccharide preparation comprisesoligosaccharides with 1, 2, 3, or 4 different species of monosaccharidessubunits. In some embodiments, the oligosaccharide preparation comprisesoligosaccharides with 1, 2, or 3 different species of monosaccharidessubunits. In some embodiments, the oligosaccharide preparation comprisesoligosaccharides with 3 different species of monosaccharides subunits.In some embodiments, the oligosaccharide preparation comprisesoligosaccharides with 2 different species of monosaccharides subunits.In some embodiments, the oligosaccharide preparation comprises onespecies of monosaccharides subunits.

In some embodiments, the oligosaccharide preparation comprises differentspecies of oligosaccharides that each oligosaccharide moleculeindependently comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 differentspecies of monosaccharides subunits. In some embodiments, a hereindescribed oligosaccharide preparation comprises 10², 10³, 10⁴, 10⁵, ormore different species of oligosaccharides. In some embodiments, some ofthe oligosaccharides in the preparation comprise one species ofmonosaccharide subunits and some other oligosaccharides in the samepreparation comprise two or more species of monosaccharides subunits.For instance, in some embodiments, when the feed sugars are glucose andgalactose, the oligosaccharide preparation can comprise oligosaccharidesthat comprise only glucose subunits, oligosaccharides that comprise onlygalactose subunits, oligosaccharides that comprise both glucose andgalactose subunits at various ratios, or any combination thereof.

In some embodiments, any or all of the n fractions of theoligosaccharide preparation comprises different species ofoligosaccharides subunits that each oligosaccharide independentlycomprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different species ofmonosaccharides subunits. In some embodiments, some of theoligosaccharides in a fraction of the preparation comprise one speciesof monosaccharide subunits and some other oligosaccharides in the samefraction of the preparation comprise two or more species ofmonosaccharides subunits.

In some embodiments, a herein described oligosaccharide preparationcomprises one or more monosaccharide subunits selected from a groupconsisting of: triose, tetrose, pentose, hexose, heptose, and anycombination thereof, wherein each of the said triose, tetrose, pentose,hexose, or heptose subunit is independently and optionallyfunctionalized and/or replaced with one of its correspondinganhydro-subunits. In some embodiments, the corresponding anhydro-subunitis a product of thermal dehydration of the monosaccharide subunit. Insome embodiments, the corresponding anhydro-subunit is a caramelizationproduct of the monosaccharide subunit.

In some embodiments, a herein described oligosaccharide preparationcomprises pentose subunits, hexose subunits, or any combination thereof,wherein each of the said pentose or hexose subunit is independently andoptionally functionalized and/or replaced with one of its correspondinganhydro-subunits. In some embodiments, the oligosaccharide preparationcomprises hexose subunits, wherein each of the said hexose subunits isindependently and optionally replaced with one of its correspondinganhydro-subunits.

As used herein, a tetrose refers to a monosaccharide with four carbonatoms, such as erythrose, threose, and erythrulose. As used herein, apentose refers to a monosaccharide with five carbon atoms, such asarabinose, lyxose, ribose, and xylose. As used herein, a hexose refersto a monosaccharide with six carbon atoms, such as allose, altrose,glucose, mannose, gulose, idose, galactose, talose, psicose, fructose,sorbose, and tagatose. As used herein, a heptose refers to amonosaccharide with seven carbon atoms, such as sedoheptulose andmannoheptulose.

In some embodiments, a herein described oligosaccharide preparationcomprises glucose subunit, wherein at least one glucose subunit isoptionally replaced with an anhydro-glucose subunit. In someembodiments, a herein described oligosaccharide preparation comprisesgalactose subunit, wherein at least one galactose subunit is optionallyreplaced with anhydro-galactose subunit. In some embodiments, a hereindescribed oligosaccharide preparation comprises galactose and glucosesubunits, wherein at least one galactose subunit or at least one glucosesubunit is optionally replaced with one of its correspondinganhydro-subunits. In some embodiments, a herein describedoligosaccharide preparation comprises fructose and glucose subunits,wherein at least one fructose subunit or at least one glucose subunit isoptionally replaced with one of its corresponding anhydro-subunits. Insome embodiments, a herein described oligosaccharide preparationcomprises mannose and glucose subunit, wherein at least one mannosesubunit or at least one glucose subunit is optionally replaced with oneof its corresponding anhydro-subunits.

In some embodiments, a herein described oligosaccharide preparationcomprises a gluco-galactose-oligosaccharide preparation, agluco-oligosaccharide preparation, a galacto-oligosaccharidepreparation, a fructo-oligosaccharide preparation, amanno-oligosaccharide preparation, an arabino-oligosaccharidepreparation, a xylo-oligosaccharide preparation, agluco-fructo-oligosaccharide preparation, a gluco-manno-oligosaccharidepreparation, a gluco-arabino-oligosaccharide preparation, agluco-xylo-oligosaccharide preparation, a galacto-fructo-oligosaccharidepreparation, a galacto-manno-oligosaccharide preparation, agalacto-arabino-oligosaccharide preparation, agalacto-xylo-oligosaccharide preparation, a fructo-manno-oligosaccharidepreparation, a fructo-arabino-oligosaccharide preparation, afructo-xylo-oligosaccharide preparation, a manno-arabino-oligosaccharidepreparation, a manno-xylo-oligosaccharide preparation, anarabino-xylo-oligosaccharide preparation, agalacto-arabino-xylo-oligosaccharide preparation, afructo-galacto-xylo-oligosaccharide preparation, anarabino-fructo-manno-xylo-oligosaccharide preparation, agluco-fructo-galacto-arabino-oligosaccharide preparation, afructo-gluco-arabino-manno-xylo oligosaccharide preparation, agluco-galacto-fructo-manno-arabinoxylo-oligosaccharide preparation, orany combinations thereof; wherein each of the monosaccharide subunitwithin the preparation is independently and optionally functionalizedand/or replaced with one of its corresponding anhydro-subunits.

In certain embodiments, a herein described oligosaccharide preparationcomprises more than 99% of glucose subunits by weight. In someembodiments, the oligosaccharide preparation comprises only glucosesubunits.

In some embodiments, a herein described oligosaccharide preparationcomprises about 45% to 55% of glucose subunits and about 55% to 45% ofgalactose subunits by weight. In some specific embodiments, theoligosaccharide preparation comprises about 50% glucose and 50%galactose subunits by weight.

In some embodiments, a herein described oligosaccharide preparationcomprises about 80% to 95% of glucose subunits and about 20% to 5% ofmannose subunits by weight. In some embodiments, the oligosaccharidepreparation comprises about 85% to 90% of glucose subunits and about 15%to 10% of mannose subunits by weight.

In some embodiments, a herein described oligosaccharide preparationcomprises about 80% to 95% of glucose subunits and about 20% to 5% ofgalactose subunits by weight. In some embodiments, the oligosaccharidepreparation comprises about 85% to 90% of glucose subunits and about 15%to 10% of galactose subunits by weight.

In some embodiments, a herein described oligosaccharide preparationcomprises about 80% to 95% of glucose subunits, 0% to 8% of galactosesubunits, and 5% to 20% of mannose subunits by weight. In someembodiments, the oligosaccharide preparation comprises about 80% to 90%of glucose subunits, 1% to 5% of galactose subunits, and 10% to 15% ofmannose subunits by weight.

In some embodiments, an oligosaccharide preparation described hereincomprises from about 1 wt % to about 100 wt %, from about 50 wt % toabout 100 wt %, from about 80 wt % to about 98 wt %, or from about 85 wt% to about 95 wt % of glucose subunits, or any ranges therebetween. Insome embodiments, galactose subunits are present in an oligosaccharidepreparation described herein at an amount of from about 0 wt % to about90 wt %, from about 1 wt % to about 50 wt %, from about 2 wt % to about20 wt %, or from about 5 wt % to about 15 wt %, or any rangestherebetween. In some embodiments, mannose subunits are present in anoligosaccharide preparation described herein at an amount of from about0 wt % to about 90 wt %, from about 1 wt % to about 50 wt %, from about2 wt % to about 20 wt %, or from about 5 wt % to about 15 wt %, or anyranges therebetween.

In some embodiments, a herein described oligosaccharide preparation hasa composition of monosaccharide subunits as shown in Table 28.

TABLE 28 Exemplary Compositions of Oligosaccharide Preparations Glucoseand Galactose and Mannose and Fructose and anhydro- anhydro- anhydro-anhydro- Oligo glucose galactose mannose fructose Composition subunitssubunits subunits subunits No. (wt %) (wt %) (wt %) (wt %) 1 87.5 12.5 00 2 100 0 0 0 3 85 2.5 12.5 0 4 87.5 0 12.5 0 5 50 50 0 0 6 75 0 25 0 79 6 0 0 8 90 0 10 0 9 95 5 0 0 10 97.5 2.5 0 0 11 85 5 10 0 12 85 1.513.5 0 13 80 10 10 0 14 85 0 15 0 15 85 15 0 0 16 87.5 0 0 12.5H D- vs. L-Form

In some embodiments, at least one monosaccharide subunit in anoligosaccharide is in L-form. In some embodiments, at least onemonosaccharides subunit in an oligosaccharide is in D-form. In someembodiments, the monosaccharide subunits in a herein describedoligosaccharide preparation are in their naturally-abundant form, forexample, D-glucose, D-xylose, and L-arabinose.

In some embodiments, a herein described oligosaccharide preparationcomprises a mixture of L- and D-forms of monosaccharide subunits. Insome embodiments, the ratio of monosaccharide subunits in L- to D- or inD- to L-form is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5,about 1:6, about 1:7, about 1:8, about 1:9, about 1:10, about 1:12,about 1:14, about 1:16, about 1:18, about 1:20, about 1:25, about 1:30,about 1:35, about 1:40, about 1:45, about 1:50, about 1:55, about 1:60,about 1:65, about 1:70, about 1:75, about 1:80, about 1:85, about 1:90,about 1:100 or about 1:150.

I. Functionalized Oligosaccharides

In some embodiments, one or more oligosaccharides in the preparation areindependently functionalized. Functionalized oligosaccharides may beproduced by, for example, combining one or more sugars with one or morefunctionalizing compounds in the presence of a catalyst. Methods ofproducing functionalized oligosaccharides are described in WO2012/118767, WO 2014/031956, and WO/2016/122887, which are herebyincorporated by reference in their entirety and for their disclosure.

In some embodiments, the functionalizing compound comprises one or moreacid groups (e.g., —COOH), hydroxyl groups, or N-containing groups(e.g., —CN, —NO₂, and —N(R_(a))₂, wherein R_(a) is hydrogen, alkyl,alkenyl, alkynyl, haloalkyl, heteroalkyl, cycloalkyl, aryl,heterocycloalkyl, or heteroaryl groups), S-containing groups (e.g.,thiol and sulfates), halides (e.g., —Cl), P-containing groups (e.g.,phosphate), or any combination thereof. In some embodiments, thefunctionalizing compound is linked to at least one monosaccharidesubunit via an ether, ester, oxygen-sulfur, amine, or oxygen-phosphorousbond. In some embodiments, one or more functionalizing compounds arelinked to a monosaccharide subunit via a single linkage. In someembodiments, at least one functionalizing compound is linked to one ortwo oligosaccharides via two or more linkages.

It is to be understood that for each oligosaccharide in theoligosaccharide preparation, each of the described embodiments isindependent and can be combined as if each and every combination werelisted separately; thus, any combination of the embodiments isencompassed by the present disclosure. For instance, the variousembodiments can be grouped into several categories that include but arenot limited to (i) the presence or absence of anhydro-subunit; (ii) thenumber and level of anhydro-subunit, (iii) the type of species ofanhydro-subunit, (iv) the location of anhydro-subunit, (v) the degree ofpolymerization, (vi) the molecular weight, (vii) the presence or absenceof any functional groups, (viii) the type of the oligosaccharide, (ix)the type of glycosidic linkage, and (x) the L- versus D-form.Accordingly, the described oligosaccharide preparation comprises aplurality of oligosaccharides of different species. In some embodiments,a herein described oligosaccharide preparation comprises at least 10,10², 10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ differentoligosaccharide species. In some embodiments, the preparation comprisesat least 10³, 10⁴, 10⁵, 10⁶, or 10⁹ different oligosaccharide species.In some embodiments, the preparation comprises at least 10³ differentoligosaccharide species.

III. Methods of Manufacturing Oligosaccharide Preparations

In one aspect, provided herein are methods of manufacturingoligosaccharide preparations. In some embodiments, provided herein aremethods of manufacturing oligosaccharide preparations suitable for usein a nutritional composition, such as an animal feed composition, orbeing fed directly to an animal. In one aspect, provided herein aremethods of manufacturing an oligosaccharide preparation comprisingheating an aqueous composition comprising one or more feed sugars and acatalyst to a temperature and for a time sufficient to inducepolymerization, wherein the catalyst is selected from the groupconsisting of: (+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid;3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate;α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid;butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid;methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid;tert-butylphosphonic acid; SS)-VAPOL hydrogenphosphate;6-quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate;2-(2-pyridinyl)ethanesulfonic acid;3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acidmonosodium salt hydrate; 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate;bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid;L-cysteic acid monohydrate; poly(styrene sulfonicacid-co-divinylbenzene); lysine; Ethanedisulfonic acid; Ethanesulfonicacid; Isethionic acid; Homocysteic acid; HEPBS(N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)); HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid; Methanesulfonic acid; Methaniazide;Naphthalene-1-sulfonic acid; Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid; 6-sulfoquinovose; Triflic acid;2-aminoethanesulfonic acid; Benzoic acid; Chloroacetic acid;Trifluoroacetic acid; Caproic acid; Enanthic acid; Caprylic acid;Pelargonic acid; Lauric acid; Pamitic acid; Stearic acid; Arachidicacid; Aspartic acid; Glutamic acid; Serine; Threonine; Glutamine;Cysteine; Glycine; Proline; Alanine; Valine; Isoleucine; Leucine;Methionine; Phenylalanine; Tyrosine; Tryptophan, and wherein theoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 (DP1 fraction) to n (DPn fraction), wherein n is aninteger greater than 2.

In some embodiments, n is an integer greater than or equal to 3. In someembodiments, n is an integer within a range of 1 to 100, such as 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 40, or 50. In some embodiments, thepolymerization of the feed sugars is achieved by a step-growthpolymerization. In some embodiments, the polymerization of the feedsugars is achieved by polycondensation.

A. Feed Sugar

In some embodiments, a method of manufacturing oligosaccharidepreparations described herein comprises heating one or more types offeed sugars. In some embodiments, the one or more types of feed sugarscomprise monosaccharides, disaccharides, trisaccharides,tetrasaccharides, or any mixtures thereof.

In some embodiments, the one or more feed sugars comprise glucose. Insome embodiments, the one or more feed sugars comprise glucose andgalactose. In some embodiments, the one or more feed sugars compriseglucose, xylose, and galactose. In some embodiments, the one or morefeed sugars comprise glucose and mannose. In some embodiments, the oneor more feed sugars comprise glucose and fructose. In some embodiments,the one or more feed sugars comprise glucose, fructose, and galactose.In some embodiments, the one or more feed sugars comprise glucose,galactose, and mannose.

In some embodiments, the one or more feed sugars comprise disaccharidessuch as lactose, sucrose and cellobiose. In some embodiments, the one ormore feed sugars comprise trisaccharides, such as maltotriose orraffinose. In certain embodiments, the one or more feed sugar comprisesglucose, mannose, galactose, xylose, malto-dextrin, arabinose, orgalactose, or any combinations thereof. In certain embodiments, the oneor more feed sugars comprise sugar syrup such as corn syrup. In someembodiments, the one or more feed sugars comprise glucose and lactose.In some embodiments, the one or more feed sugars comprise glucose andsucrose.

In some embodiments, the type of feed sugars can impact the resultingmanufactured oligosaccharide preparations. For example, in somevariations where the one or more feed sugars are all glucose, theresulting oligosaccharide preparations comprise gluco-oligosaccharidespreparations. In other embodiments, where the one or more feed sugarsare all mannose, the resulting oligosaccharide preparations comprisemanno-oligosaccharide preparations. In some embodiments, wherein the oneor more feed sugars comprise glucose and galactose, the resultingoligosaccharide preparations comprise gluco-galacto-oligosaccharidepreparations. In yet other embodiments, where the one or more feedsugars comprise xylose, glucose and galactose, the resultingoligosaccharide preparations comprise gluco-galacto-xylo-oligosaccharidepreparations.

In some embodiments, each of the one or more feed sugars can beindependently in its de-hydrate or hydrate form. In some embodiments,the one or more feed sugars comprise glucose, galactose, fructose,mannose, or any combination thereof, and wherein each of the glucose,galactose, fructose, or mannose is independently in its mono-hydrate orde-hydrate form. In some embodiments, the one or more feed sugarscomprise a monosaccharide mono-hydrate such as glucose monohydrate. Insome embodiments, the one or more feed sugars comprise a saccharidedi-hydrate such as trehalose di-hydrate. In some embodiments, the one ormore feed sugars comprise at least one sugar in its de-hydrate form andat least one sugar in its hydrate form.

In some embodiments, the one or more feed sugars can be provided as asugar solution, in which the sugars are combined with water and fed intothe reactor. In some embodiments, the sugars can be fed into the reactorin a solid form and combined with water in the reactor. In someembodiments, the one or more feed sugars are combined and mixed beforethe addition of water. In other embodiments, the one or more feed sugarsare combined into water and mixed thereafter.

In some embodiments, the method comprises combining two or more feedsugars with the catalyst to produce an oligosaccharide preparation. Insome embodiments, the two or more feed sugars comprise from glucose,galactose, fructose, mannose, lactose, or any combination thereof. Insome embodiments, the method comprises combining a mixture of sugars(e.g., monosaccharides, disaccharides, and/or trisaccharides) with thecatalyst to produce an oligosaccharide preparation. In otherembodiments, the method comprises combining a mixture of sugars andsugar alcohols with the catalyst to produce an oligosaccharidepreparation.

In some embodiments, the one or more feed sugars comprise functionalizedor modified sugars. Functionalized or modified sugars may comprise aminosugars, sugar acids, sugar alcohols, sugar amides, sugar ethers, or anycombination thereof. In some embodiments, amino sugars refer to sugarmolecules in which a hydroxyl group is replaced with an amine group.Exemplary amino sugars include, but are not limited to,N-Acetyl-d-glucosamine, mannosamine, neuraminic acid, muramic acid,N-acetyl-neuramin, N-acetyl-muramic, N-acetyl-galactosamine,N-acetyl-mannosa, N-glycolylneuram, acarviosin, D-glucosamine, andD-galactosamine.

In embodiments, sugar acids refer to sugars with a carboxyl group.Exemplary sugar acids include, but are not limited to, aldonic acids(such as glyceric acid, xylonic acid, gluconic acid, and ascorbic acid),ulosonic acids (such as neuraminic acid and ketodeoxyoctulosonic acid),uronic acids (such as glucuronic acid, galacturonic acid, and iduronicacid), and aldaric acids (such as tartaric acid, mucic acid, andsaccharic acid).

In some embodiments, sugar alcohols refer to sugar-derived polyols.Exemplary sugar alcohols include, but are not limited to, ethyleneglycol, arabitol, glycerol, erythritol, threitol, xylitol, ribitol,mannitol, sorbitol, galactitol, fucitol, iditol, inositol, andvolemitol.

In some embodiments, sugar amides refer to sugar molecules that containa —C(═O)—N— group. In embodiments, sugar ethers refer to sugar moleculesthat contain an ether bond, such as glucosides.

In some embodiments, the functionalized or modified sugars compriseglucosamine, N-acetylglucosamine, glucuronic acid, galacturonic acid,glucitol, xylitol, mannitol, sorbitol. In some embodiments, the one ofmore feed sugars comprise deoxysugars, such as fucose, rhamnose,deoxyribose, or fuculose.

In some embodiments, a herein described method of manufacturingoligosaccharide preparation is performed at gram scale. In someembodiments, a herein described method of manufacturing oligosaccharidepreparation is performed at kilogram or higher scale. Accordingly, insome embodiments, the method comprises heating an aqueous compositioncomprising one or more feed sugars at a quantity of more than 0.5, morethan 1, more than 2, more than 3, more than 4, more than 5, more than 6,more than 7, more than 9, more than 10, more than 100, or more than 1000kg. In some embodiments, the method comprises heating an aqueouscomposition comprising one or more feed sugars at a quantity of no morethan 0.5, 1, 2, 3, 4, 5, 6, 7, 9, 10, 100, 1000, or 1500 kg. In someembodiments, the method comprises heating an aqueous compositioncomprising one or more feed sugars at a quantity of more than 1 kg.

B. Catalyst

In some embodiments, the catalyst provided herein comprises one or moreacids. In some embodiments, the catalyst provided herein comprisesmineral acid, carboxylic acid; amino acid; sulfonic acid; boronic acid;phosphonic acid; phosphinic acid; sulfuric acid; phosphoric acid;poly(styrene sulfonic acid-co-vinylbenzyl-imidazoliumsulfate-co-divinylbenzene); poly(styrene sulfonicacid-co-divinylbenzene); (+)-camphor-10-sulfonic acid;2-pyridinesulfonic acid; 3-pyridinesulfonic acid;8-hydroxy-5-quinolinesulfonic acid hydrate;α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid;butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid;methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid;tert-butylphosphonic acid; SS)-VAPOL hydrogenphosphate;6-quinolinesulfonic acid; 3-(1-pyridinio)-1-propanesulfonate;2-(2-pyridinyl)ethanesulfonic acid;3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acidmonosodium salt hydrate; 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate;bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid;L-cysteic acid monohydrate; acetic acid; propionic acid; butanoic acid;glutamic acid; lysine; Ethanedisulfonic acid; Ethanesulfonic acid;Isethionic acid; Homocysteic acid; HEPBS(N-(2-Hydroxyethyl)piperazine-N′-(4-butane sulfonic acid)); HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid; Methanesulfonic acid; Methaniazide;Naphthalene-1-sulfonic acid; Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid; 6-sulfoquinovose; Triflic acid;2-aminoethanesulfonic acid; Benzoic acid; Chloroacetic acid;Trifluoroacetic acid; Caproic acid; Enanthic acid; Caprylic acid;Pelargonic acid; Lauric acid; Pamitic acid; Stearic acid; Arachidicacid; Aspartic acid; Glutamic acid; Serine; Threonine; Glutamine;Cysteine; Glycine; Proline; Alanine; Valine; Isoleucine; Leucine;Methionine; Phenylalanine; Tyrosine; Tryptophan; polymeric acid;carbon-supported acid; or any combination thereof.

In some embodiments, the catalyst provided herein comprises:(+)-camphor-10-sulfonic acid; 2-pyridinesulfonic acid;3-pyridinesulfonic acid; 8-hydroxy-5-quinolinesulfonic acid hydrate;α-hydroxy-2-pyridinemethanesulfonic acid; (β)-camphor-10-sulfonic acid;butylphosphonic acid; diphenylphosphinic acid; hexylphosphonic acid;methylphosphonic acid; phenylphosphinic acid; phenylphosphonic acid;tert-butylphosphonic acid; SS)-VAPOL hydrogenphosphate;6-quinolinesulfonic acid, 3-(1-pyridinio)-1-propanesulfonate;2-(2-pyridinyl)ethanesulfonic acid;3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acidmonosodium salt hydrate; 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate;bis(4-methoxyphenyl)phosphinic acid; phenyl(3,5-xylyl)phosphinic acid;L-cysteic acid monohydrate; poly(styrene sulfonicacid-co-divinylbenzene); lysine; Ethanedisulfonic acid; Ethanesulfonicacid; Isethionic acid; Homocysteic acid; HEPBS(N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)); HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid);2-Hydroxy-3-morpholinopropanesulfonic acid;2-(N-morpholino)ethanesulfonic acid; Methanesulfonic acid; Methaniazide;Naphthalene-1-sulfonic acid; Naphthalene-2-sulfonic acid;Perfluorobutanesulfonic acid; 6-sulfoquinovose; Triflic acid;2-aminoethanesulfonic acid; Benzoic acid; Chloroacetic acid;Trifluoroacetic acid; Caproic acid; Enanthic acid; Caprylic acid;Pelargonic acid; Lauric acid; Pamitic acid; Stearic acid; Arachidicacid; Aspartic acid; Glutamic acid; Serine; Threonine; Glutamine;Cysteine; Glycine; Proline; Alanine; Valine; Isoleucine; Leucine;Methionine; Phenylalanine; Tyrosine; Tryptophan; or any combinationthereof.

In some embodiments, the catalyst provided herein is(+)-camphor-10-sulfonic acid. In some embodiments, the catalyst providedherein is 2-pyridinesulfonic acid. In some embodiments, the catalystprovided herein is 3-pyridinesulfonic acid. In some embodiments, thecatalyst provided herein is 8-hydroxy-5-quinolinesulfonic acid hydrate.In some embodiments, the catalyst provided herein isα-hydroxy-2-pyridinemethanesulfonic acid. In some embodiments, thecatalyst provided herein is ((3)-camphor-10-sulfonic acid. In someembodiments, the catalyst provided herein is butylphosphonic acid. Insome embodiments, the catalyst provided herein is diphenylphosphinicacid. In some embodiments, the catalyst provided herein ishexylphosphonic acid. In some embodiments, the catalyst provided hereinis methylphosphonic acid. In some embodiments, the catalyst providedherein is phenylphosphinic acid. In some embodiments, the catalystprovided herein is phenylphosphonic acid. In some embodiments, thecatalyst provided herein is tert-butylphosphonic acid. In someembodiments, the catalyst provided herein is SS)-VAPOLhydrogenphosphate. In some embodiments, the catalyst provided herein is6-quinolinesulfonic acid. In some embodiments, the catalyst providedherein is 3-(1-pyridinio)-1-propanesulfonate. In some embodiments, thecatalyst provided herein is 2-(2-pyridinyl)ethanesulfonic acid. In someembodiments, the catalyst provided herein is3-(2-pyridyl)-5,6-diphenyl-1,2,4-triazine-p,p′-disulfonic acidmonosodium salt hydrate. In some embodiments, the catalyst providedherein is 1,1′-binaphthyl-2,2′-diyl-hydrogenphosphate. In someembodiments, the catalyst provided herein isbis(4-methoxyphenyl)phosphinic acid. In some embodiments, the catalystprovided herein is phenyl(3,5-xylyl)phosphinic acid. In someembodiments, the catalyst provided herein is L-cysteic acid monohydrate.In some embodiments, the catalyst provided herein is poly(styrenesulfonic acid-co-divinylbenzene). In some embodiments, the catalystprovided herein is lysine.

In some embodiments, the catalyst is Ethanedisulfonic acid. In someembodiments, the catalyst is Ethanesulfonic acid. In some embodiments,the catalyst is Isethionic acid. In some embodiments, the catalyst isHomocysteic acid. In some embodiments, the catalyst is HEPBS(N-(2-Hydroxyethyl)piperazine-N′-(4-butanesulfonic acid)). In someembodiments, the catalyst is HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). In someembodiments, the catalyst is 2-Hydroxy-3-morpholinopropanesulfonic acid.In some embodiments, the catalyst is 2-(N-morpholino) ethanesulfonicacid. In some embodiments, the catalyst is Methanesulfonic acid. Inembodiments, the catalyst is Naphthalene-1-sulfonic acid. In someembodiments, the catalyst is some embodiments, the catalyst isMethaniazide. In some Naphthalene-2-sulfonic acid. In some embodiments,the catalyst is Perfluorobutanesulfonic acid. In some embodiments, thecatalyst is 6-sulfoquinovose. In some embodiments, the catalyst isTriflic acid. In some embodiments, the catalyst is 2-aminoethanesulfonicacid. In some embodiments, the catalyst is Benzoic acid. In someembodiments, the catalyst is Chloroacetic acid. In some embodiments, thecatalyst is Trifluoroacetic acid. In some embodiments, the catalyst isCaproic acid. In some embodiments, the catalyst is Enanthic acid. Insome embodiments, the catalyst is Caprylic acid. In some embodiments,the catalyst is Pelargonic acid. In some embodiments, the catalyst isLauric acid. In some embodiments, the catalyst is Pamitic acid. In someembodiments, the catalyst is Stearic acid. In some embodiments, thecatalyst is Arachidic acid. In some embodiments, the catalyst isAspartic acid. In some embodiments, the catalyst is Glutamic acid. Insome embodiments, the catalyst is Serine. In some embodiments, thecatalyst is Threonine. In some embodiments, the catalyst is Glutamine.In some embodiments, the catalyst is Cysteine. In some embodiments, thecatalyst is Glycine. In some embodiments, the catalyst is Proline. Insome embodiments, the catalyst is Alanine. In some embodiments, thecatalyst is Valine. In some embodiments, the catalyst is Isoleucine. Insome embodiments, the catalyst is Leucine. In some embodiments, thecatalyst is Methionine. In some embodiments, the catalyst isPhenylalanine. In some embodiments, the catalyst is Tyrosine. In someembodiments, the catalyst is Tryptophan.

In some embodiments, the catalyst provided herein is a polymericcatalyst or a carbon-supported catalyst disclosed in WO 2016122887,which is hereby incorporated by reference in its entirety and for itsdisclosure.

In some embodiments, the catalyst provided herein is present in anamount of from about 0.01% to about 5%, from about 0.02% to about 4%,from about 0.03% to about 3%, or from about 0.05% to about 2% of the oneor more feed sugars by dry weight. In some embodiments, the catalystprovided herein is present in an amount of from about 1% to 2% of theone or more feed sugars by dry weight. In some embodiments, the catalystprovided herein is present in an amount of about 0.5%, about 0.6%, about0.7%, about 0.8%, about 0.9%, about 1.0%, about 1.1%, about 1.2%, about1.3%, about 1.4%, about 1.5%, about 1.6%, about 1.7%, about 1.8%, about1.9%, about 2.0%, about 2.1%, about 2.2%, about 2.3%, about 2.4%, about2.5%, about 2.6%, about 2.7%, about 2.8%, about 2.9%, or about 3.0% ofthe one or more feed sugars by dry weight.

In some embodiments, the catalyst provided herein is present in anamount of from about 0.01% to about 5%, from about 0.02% to about 4%,from about 0.03% to about 3%, or from about 0.05% to about 2% of theaqueous composition by dry weight. In some embodiments, the catalystprovided herein is present in an amount of from about 1% to 2% of theaqueous composition by dry weight. In some embodiments, the catalystprovided herein is present in an amount of about 0.8%, about 0.9%, about1.0%, about 1.1%, about 1.2%, about 1.3%, about 1.4%, about 1.5%, about1.6%, about 1.7%, about 1.8%, about 1.9%, about 2.0%, about 2.1%, about2.2%, about 2.3%, about 2.4%, about 2.5%, about 2.6%, about 2.7%, about2.8%, about 2.9%, or about 3.0% of the aqueous composition by dryweight.

In some embodiments, the catalyst provided herein is a combination oftwo or more different catalysts. In some embodiments, the catalystcomprises a recyclable catalyst such as resins and polymeric catalystsand a non-recyclable catalyst. In some embodiments, where the catalystcomprises at least two different catalysts, each of the catalyst ispresent in an amount provided herein. In other embodiments, where thecatalyst comprises at least two different catalysts, the at least twodifferent catalysts are present in aggregate in an amount providedherein.

In some embodiments, the catalyst is added into the aqueous compositionin a dry form. In other embodiments, the catalyst is added into theaqueous composition in a wet form such as in an aqueous solution. Insome embodiment, the catalyst is combined with the one or more feedsugars before the addition of water. In other embodiments, the catalystis dissolved into water before its combining with the one or more feedsugars. In some embodiments, the method provided herein comprisesproducing an aqueous composition by combining the one or more feedsugars in the de-hydrate form and the catalyst in a wet form (e.g., asan aqueous solution).

C. Addition of Water

In some embodiments, the methods of manufacturing the oligosaccharidepreparations comprise adding water to form the aqueous composition. Insome embodiments, all or part of the water in the aqueous composition isadded as free water. In other embodiments, all of the water in theaqueous composition is added as bonded water, for example, in saccharidemono- or di-hydrate. In some embodiments, all of the water in theaqueous composition is added as bonded water in monosaccharidemono-hydrate, such as glucose mono-hydrate. In certain embodiments, allor part of the water in the aqueous composition is added with thecatalyst, i.e., via a catalyst solution.

D. Water Content

As the methods of manufacturing the oligosaccharide preparationsproceed, water can be produced through reaction. For example, in someembodiments, water is produced (i) with the formation of a glycosidicbond, (ii) with the formation of an anhydro-subunit, or (iii) throughother mechanisms or sources. As the sugar condensation and dehydrationreactions both involve water, in some embodiments, the water contentinfluences the composition of the oligosaccharide preparation.

Further, in some embodiments, water content influences the viscosity ofthe aqueous composition, which in turn may affect the effectiveness ofmixing of the aqueous composition. For example, in some embodiments, anoverly viscous aqueous composition can lead to an undesirableheterogeneous catalyst distribution in the aqueous composition.Moreover, in some embodiments, very low water content may lead to thesolidification of the aqueous composition, which prevents effectivemixing. On the other hand, in some other embodiments, exceedingly highwater content may impede sugar condensation reaction and lower the levelof the anhydro-subunits. Accordingly, the present disclosure describessuitable water content for the manufacturing of oligosaccharidepreparations.

In some embodiments, a herein described method of manufacturingoligosaccharide preparation comprises forming and/or heating an aqueouscomposition. In some embodiments, the aqueous composition comprises fromabout 0% to about 80%, from about 0% to about 70%, from about 0% toabout 60%, from about 0% to about 50%, from about 0% to about 40%, fromabout 0% to about 35%, from about 0% to about 30%, from about 0% toabout 25%, from about 0% to about 20%, from about 0% to about 19%, fromabout 0% to about 18%, from about 0% to about 17%, from about 0% toabout 16%, from about 0% to about 15%, from about 0% to about 14%, fromabout 0% to about 13%, from about 0% to about 12%, from about 0% toabout 11%, from about 0% to about 10%, from about 0% to about 9%, fromabout 0% to about 8%, from about 0% to about 7%, from about 0% to about6%, from about 0% to about 5%, from about 0% to about 4%, from about 0%to about 3%, from about 0% to about 2%, or from about 0% to about 1% ofwater by total weight. In some embodiments, the aqueous compositioncomprises from about 1% to about 20%, from about 1% to about 18%, fromabout 1% to about 16%, from about 1% to about 14%, from about 1% toabout 12%, from about 1% to about 10%, from about 1% to about 8%, fromabout 1% to about 6%, or from about 1% to about 4% of water by totalweight. In some embodiments, the aqueous composition comprises fromabout 3% to about 16%, from about 3% to about 14%, from about 3% toabout 12%, from about 3% to about 10%, from about 3% to about 8%, fromabout 3% to about 6%, from about 5% to about 16%, from about 5% to about14%, from about 5% to about 12%, from about 5% to about 10%, from about7% to about 16%, from about 7% to about 14%, from about 7% to about 12%,from about 7% to about 10%, or from about 8% to about 10% of water bytotal weight. In some embodiments, the aqueous composition comprisesabout 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%,about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about14%, or about 15% of water by total weight. In some embodiments, theaqueous composition comprises about 9% water by total weight. It shouldbe understood, however, that the amount of water in the aqueouscomposition can be adjusted based on the reaction conditions andspecific catalyst used. In some embodiments, the water content in theaqueous composition as disclosed above is measured at the beginning ofthe reaction, for example, before heating the feed sugars. In someembodiments, the water content in the aqueous composition as disclosedabove is measured at the end of the polymerization or condensationreaction. In some embodiments, the water content in the aqueouscomposition as disclosed above is measured as an average water contentof the beginning of the reaction and at the end of the reaction.

In certain embodiments, a method described herein can further comprisemonitoring the content of water present in the aqueous compositionand/or the ratio of water to sugars or catalyst over a period of time.In some embodiments, the method further comprises removing at least aportion of water in the aqueous composition, for example, bydistillation. Any method known in the art can be used to remove waterfrom the aqueous composition, including, for example, by vacuumfiltration, vacuum distillation, heating, steam, hot air, and/orevaporation.

In some embodiments, herein described oligosaccharide preparations arehygroscopic. Thus, in some embodiments, the hygroscopicity of the feedsugars and the oligosaccharides formed in the polymerization can affectthe rate by which the water can be removed from the aqueous composition.

In some embodiments, a herein described method comprises removing atleast a portion of water in the aqueous composition such that the watercontent in the aqueous composition is from about 1% to about 20%, fromabout 1% to about 18%, from about 1% to about 16%, from about 1% toabout 14%, from about 1% to about 12%, from about 1% to about 10%, fromabout 1% to about 8%, from about 2% to about 16%, from about 2% to about14%, from about 2% to about 12%, from about 2% to about 10%, from about2% to about 8%, from about 2% to about 6%, from about 4% to about 16%,from about 4% to about 14%, from about 4% to about 12%, from about 4% toabout 10%, from about 4% to about 8%, from about 6% to about 16%, fromabout 6% to about 12%, from about 6% to about 10%, or from about 6% toabout 8% by total weight. In some embodiments, the method comprisesremoving at least a portion of water in the aqueous composition suchthat the water content in the aqueous composition is from about 2% toabout 10%, from about 2% to about 8%, or from about 4% to about 8% bytotal weight. In some embodiments, the method comprises removing atleast a portion of water in the aqueous composition such that the watercontent in the aqueous composition is about 2%, about 3%, about 4%,about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% by totalweight. In some embodiments, the method comprises removing at least aportion of water in the aqueous composition such that the water contentin the aqueous composition is from about 4% to about 8% by total weight.In some embodiments, the method comprises removing at least a portion ofwater in the aqueous composition such that, at the end of thepolymerization and/or condensation reaction, the water content in theaqueous composition is a water content as disclosed above. In someembodiments, the method comprises removing at least a portion of waterin the aqueous composition such that, at the beginning of thepolymerization and/or condensation reaction, the water content in theaqueous composition is a water content as disclosed above. In someembodiments, the method comprises removing at least a portion of waterin the aqueous composition such that, the average water content in theaqueous composition at the beginning and the end of the polymerizationand/or condensation reaction is within a range as disclosed above. Insome embodiments, the method comprises removing at least a portion ofwater in the aqueous composition such that, throughout thepolymerization and/or condensation reaction, the water content in theaqueous composition remains within a range as disclosed above.

In some embodiments, a herein described method comprises adding at leasta portion of water in the aqueous composition such that the watercontent in the aqueous composition is from about 1% to about 20%, fromabout 1% to about 18%, from about 1% to about 16%, from about 1% toabout 14%, from about 1% to about 12%, from about 1% to about 10%, fromabout 1% to about 8%, from about 2% to about 16%, from about 2% to about14%, from about 2% to about 12%, from about 2% to about 10%, from about2% to about 8%, from about 2% to about 6%, from about 4% to about 16%,from about 4% to about 14%, from about 4% to about 12%, from about 4% toabout 10%, from about 4% to about 8%, from about 6% to about 16%, fromabout 6% to about 12%, from about 6% to about 10%, or from about 6% toabout 8% by total weight. In some embodiments, the method comprisesadding at least a portion of water in the aqueous composition such thatthe water content in the aqueous composition is from about 2% to about10%, from about 2% to about 8%, or from about 4% to about 8% by totalweight. In some embodiments, the method comprises adding at least aportion of water in the aqueous composition such that the water contentin the aqueous is about 2%, about 3%, about 4%, about 5%, about 6%,about 7%, about 8%, about 9%, or about 10% by total weight. In someembodiments, the method comprises adding at least a portion of water inthe aqueous composition such that the water content in the aqueouscomposition is from about 4% to about 8% by total weight. In someembodiments, the method comprises adding at least a portion of water inthe aqueous composition such that, at the end of the polymerizationand/or condensation reaction, the water content in the aqueouscomposition is a water content as disclosed above. In some embodiments,the method comprises adding at least a portion of water in the aqueouscomposition such that, at the beginning of the polymerization and/orcondensation reaction, the water content in the aqueous composition is awater content as disclosed above. In some embodiments, the methodcomprises adding at least a portion of water in the aqueous compositionsuch that, the average water content in the aqueous composition at thebeginning and the end of the polymerization and/or condensation reactionis within a range as disclosed above. In some embodiments, the methodcomprises adding at least a portion of water in the aqueous compositionsuch that, throughout the polymerization and/or condensation reaction,the water content in the aqueous composition remains within a range asdisclosed above.

In some embodiments, the degrees of polymerization of theoligosaccharides and/or the amount and type of the anhydro-subunitswithin the oligosaccharide preparation can be regulated by adjusting orcontrolling the content of water present in the aqueous compositionthroughout the manufacturing process. For example, in some embodiments,the degrees of polymerization of the oligosaccharides and the amount ofthe anhydro-subunits are increased by decreasing the water content.

Accordingly, in some embodiments, a herein described method comprisesin-process control (IPC) of the water content, which can comprisemonitoring water content, maintaining water content, increasing watercontent, decreasing water content, or any combination thereof. In someembodiments, an IPC process comprises maintaining the water contentwhile the aqueous composition is heated to a temperature describedherein. In some embodiments, the method comprises maintaining the watercontent for the time sufficient to induce polymerization. In someembodiments, the method comprises maintaining the water content within adisclosed range by either adding water or removing water from theaqueous composition, or both. In some embodiments, the method comprisesmaintaining the water content within a disclosed range by distillation.In some embodiments, the method comprises maintaining the water contentwithin a disclosed range by vacuum distillation. In some embodiments,the method comprises maintaining the water content within a disclosedrange by distillation under atmosphere pressure.

In some embodiments, the water content of the aqueous composition ismaintained within a range of from about 1% to about 20%, from about 1%to about 18%, from about 1% to about 16%, from about 1% to about 14%,from about 1% to about 12%, from about 1% to about 10%, from about 1% toabout 8%, from about 2% to about 16%, from about 2% to about 14%, fromabout 2% to about 12%, from about 2% to about 10%, from about 2% toabout 8%, from about 2% to about 6%, from about 4% to about 16%, fromabout 4% to about 14%, from about 4% to about 12%, from about 4% toabout 10%, from about 4% to about 8%, from about 6% to about 16%, fromabout 6% to about 12%, from about 6% to about 10%, or from about 6% toabout 8% by total weight. In some embodiments, the water content of theaqueous composition is maintained within a range of from about 2% toabout 10%, from about 2% to about 8%, or from about 4% to about 8% bytotal weight. In some embodiments, the water content of the aqueouscomposition is maintained within a range of from about 2% to about 8% bytotal weight.

The water content of the aqueous composition can be determined by avariety of analytical methods and instruments. In some embodiments, thewater content is determined by an evaporation method (e.g., loss ondrying technique), a distillation method, or a chemical reaction method(e.g., Karl Fischer titration). In some embodiments, the water contentis determined by an analytical instrument such as a moisture analyzer.In some embodiments, the water content is determined by Karl Fischertitration.

In some embodiments, the water content of the aqueous composition ismeasured during the reaction and is used to implement in-process control(IPC) of the water content. In certain embodiments, the water content ofthe reaction is measured by Karl-Fisher titration, IR spectroscopy, NIRspectroscopy, conductivity, viscosity, density, mixing torque, or mixingenergy. In some embodiments, the measurement of the water content of thereaction is used to control an apparatus that actively adjusts the watercontent of the reaction, such as a water addition pump or flow valve.

Without being bound by theory, it is believed that water content duringthe sugar polymerization and/or condensation reaction can affect thelevel of the anhydro-subunits in a herein described oligosaccharidepreparation. For example, as illustrated in FIG. 34, in someembodiments, a higher water content correlates with a lower level ofanhydro-subunits. In some embodiments, a lower reaction temperature cancorrelate with a lower level of anhydro-subunits content.

E. Temperature

In some embodiments, the degrees of polymerization of theoligosaccharides and/or the amount and type of the anhydro-subunitswithin the oligosaccharide preparation can be regulated by adjusting thetemperature, to which the aqueous composition is heated. In someembodiments, a herein described method of manufacturing anoligosaccharide preparation comprises heating the aqueous composition toa temperature of from about 80° C. to about 250° C., from about 90° C.to about 200° C., from about 100° C. to about 200° C., from about 100°C. to about 180° C., from about 110° C. to about 170° C., from about120° C. to about 160° C., from about 130° C. to about 150° C., or fromabout 135° C. to about 145° C. In some embodiments, the method ofmanufacturing an oligosaccharide preparation comprises heating theaqueous composition to a temperature of from about 100° C. to about 200°C., from about 100° C. to about 180° C., from about 110° C. to about170° C., from about 120° C. to about 160° C., from about 130° C. toabout 150° C., or from about 135° C. to about 145° C. In someembodiments, the method of manufacturing an oligosaccharide preparationcomprises heating the aqueous composition to a temperature of from about135° C. to about 145° C. In other embodiments, the method ofmanufacturing an oligosaccharide preparation comprises heating theaqueous composition to a temperature of from about 125° C. to about 135°C.

F. Reaction Time

In some embodiments, a herein described method of manufacturing anoligosaccharide preparation comprises heating the aqueous compositionfor a sufficient time. In some embodiments, the degrees ofpolymerization of the oligosaccharides manufactured according to themethods described herein can be regulated by the reaction time.

In some embodiments, the sufficient time is prescribed by a number ofhours. For example, in some embodiments, the sufficient time is at least30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, atleast 4 hours, at least 5 hours, at least 6 hours, at least 7 hour, atleast 8 hours, at least 9 hours, or at least 10 hours. In someembodiments, the sufficient time is from about 1 to about 24 hours, fromabout 1 to about 16 hours, from about 1 to about 8 hours, from about 1to about 4 hours, from about 1 to about 3 hours, from about 1 to about 2hours, from about 2 to about 12 hours, from about 2 to about 10 hours,from about 2 to about 8 hours, from about 2 to about 6 hours, from about2 to about 4 hours, from about 3 to about 8 hours, from about 3 to about6 hours, from about 3 to about 5 hours, or from about 3 to about 4hours.

In some embodiments, the sufficient time is determined by measuring oneor more chemical or physical properties of the oligosaccharidepreparation, for example, water content, viscosity, molecular weight,anhydro-subunit content, and/or the distribution of degrees ofpolymerization.

In some embodiments, the molecular weight of the oligosaccharidepreparation is monitored during polymerization. In some embodiments, themethod comprises heating the aqueous composition for a time sufficientfor the aqueous composition to reach a number average molecular weightor weight average molecular weight as described herein. In certainembodiments, the method comprises heating the aqueous composition for atime sufficient for the aqueous composition to reach a number averagemolecular weight within a range of from about 300 to about 5000 g/mol,from about 500 to about 5000 g/mol, from about 700 to about 5000 g/mol,from about 500 to about 2000 g/mol, from about 700 to about 2000 g/mol,from about 700 to about 1500 g/mol, from about 300 to about 1500 g/mol,from about 300 to about 2000 g/mol, from about 400 to about 1000 g/mol,from about 400 to about 900 g/mol, from about 400 to about 800 g/mol,from about 500 to about 900 g/mol, or from about 500 to about 800 g/mol.In certain embodiments, the method comprises heating the aqueouscomposition for a time sufficient for the aqueous composition to reach anumber average molecular weight of from about 500 to about 2000 g/mol.In certain embodiments, the method comprises heating the aqueouscomposition for a time sufficient for the aqueous composition to reach aweight average molecular weight within a range of from about 300 toabout 5000 g/mol, from about 500 to about 5000 g/mol, from about 700 toabout 5000 g/mol, from about 500 to about 2000 g/mol, from about 700 toabout 2000 g/mol, from about 700 to about 1500 g/mol, from about 300 toabout 1500 g/mol, from about 300 to about 2000 g/mol, from about 400 toabout 1300 g/mol, from about 400 to about 1200 g/mol, from about 400 toabout 1100 g/mol, from about 500 to about 1300 g/mol, from about 500 toabout 1200 g/mol, from about 500 to about 1100 g/mol, from about 600 toabout 1300 g/mol, from about 600 to about 1200 g/mol, or from about 600to about 1100 g/mol. In certain embodiments, the method comprisesheating the aqueous composition for a time sufficient for the aqueouscomposition to reach a weight average molecular weight of from about 700to about 3000 g/mol.

In some embodiments, the sufficient time is the time required for theaqueous composition to reach reaction equilibrium at the respectivereaction temperature. Accordingly, in some embodiments, the methodcomprises heating the aqueous composition for a time sufficient for theaqueous composition to reach equilibrium. For example, in someembodiments, the equilibrium is determined by measuring the molecularweight, viscosity, or DP distribution of the aqueous composition.

In certain embodiments, the equilibrium is determined by measuring thenumber average or weight average molecular weight of the aqueouscomposition. In some embodiments, the equilibrium is determined by thenumber or weight average molecular weight of the aqueous compositionthat remains essentially unchanged over time. In some embodiments, theequilibrium is determined by a change of the number or weight averagemolecular weight of the aqueous composition that is less than certainpercentage over a period of time. In some embodiments, the molecularweight of the aqueous composition is measured by HPLC or SEC.

In some embodiments, the equilibrium is determined by a change of thenumber or weight average molecular weight of the aqueous composition ofless than 25%, less than 20%, less than 15%, less than 10%, or less than5% over a period of time. In some embodiments, the equilibrium isdetermined by a change of the number or weight average molecular weightof the aqueous composition over a period of 3 hours, 2 hours, 1 hour, 30minutes, 20 minutes, or 10 minutes. In some embodiments, the equilibriumis determined by a change of the weight average molecular weight of theaqueous composition of less than 15% over the period of 1 hour.

In certain embodiments, the equilibrium is determined by measuring theviscosity of the aqueous composition. In some embodiments, theequilibrium is determined by the viscosity of the aqueous compositionthat remains essentially unchanged over time. In some embodiments, theequilibrium is determined by a change of the viscosity of the aqueouscomposition that is less than certain percentage over a period of time.In some embodiments, the viscosity of the aqueous composition ismeasured by a viscometer or rheometer.

In some embodiments, the equilibrium is determined by a change of theviscosity of the aqueous composition of less than 25%, less than 20%,less than 15%, less than 10%, or less than 5% over a period of time. Insome embodiments, the equilibrium is determined by a change of theviscosity of the aqueous composition over a period of 3 hours, 2 hours,1 hour, 30 minutes, 20 minutes, or 10 minutes. In some embodiments, theequilibrium is determined by a change of the viscosity of the aqueouscomposition of less than 15% over the period of 1 hour.

In certain embodiments, the equilibrium is determined by measuring theDP distribution of the aqueous composition. In some embodiments, theequilibrium is determined by the DP distribution of the aqueouscomposition that remains essentially unchanged over time. In someembodiments, a change of the DP distribution of the aqueous compositionis determined by calculating a series of Km, wherein

${{Km} = \frac{\left\lbrack {DP_{m}} \right\rbrack\left\lbrack {H_{2}O} \right\rbrack}{\left\lbrack {DP_{m - 1}} \right\rbrack\left\lbrack {DP1} \right\rbrack}},$

wherein [H₂O] represents the molar water concentration (mol/L), and[DP1], [DPm-₁], and [DPm] represent the molar concentrations ofoligosaccharides (mol/L) in the DP1, DPm-₁, and DPm fraction,respectively. For example, K2 equals [DP2][H₂O]/[DP1][DP1] according tothe above formula. In some embodiments, m is an integer larger than 1and less than n. In some embodiments, m is an integer larger than 1 andless than or equal to n. In some embodiments, m equals n. In someembodiments, m is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

In some embodiments, the concentration of the oligosaccharides in theDP1, DPm-1, and DPm fractions are determined by SEC, HPLC, FFF, A4F,mass spectrometry, or any other suitable method. In some embodiments,the concentration of the oligosaccharides in the DP1, DPm-1, and DPmfractions are determined by SEC such as GPC. In some embodiments, theconcentration of the oligosaccharides in the DP1, DPm-1, and DPmfractions are determined by mass spectrometry such as GC-MS, LC-MS/MS,and MALDI-MS. In some embodiments, the concentration of theoligosaccharides in the DP1, DPm-1, and DPm fractions are determined byHPLC. In some embodiments, the water concentration is determined by anevaporation method (e.g., loss on drying technique), a distillationmethod, or by a chemical reaction method (e.g., Karl Fischer titration).In some embodiments, the water concentration is determined by anysuitable analytical instrument such as a moisture analyzer.

In some embodiments, the method comprises calculating a series of atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20, at least 30, at least40, or at least 50 Km numbers. In some embodiments, the method comprisescalculating a series of at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, or at least 15 Kmnumbers. In some embodiments, the method comprises calculating about 3,4, 5, 6, 7, 8, 9, 10, or 15 Km numbers. In some embodiments, the methodcomprises calculating K2 to K4, K2 to K5, K2 to K6, K2 to K7, K2 to K8,K2 to K9, K2 to K10, K2 to K11, K2 to K12, K2 to K13, K2 to K14, K2 toK15, K3 to K5, K3 to K6, K3 to K7, K3 to K8, K3 to K9, K3 to K10, K3 toK11, K3 to K12, K3 to K13, K3 to K14, or K3 to K15. In certainembodiments, the method comprises calculating K2 to K4 or K3 to K5.

In some embodiments, the value of Km depends on the temperature, waterconcentration, and/or the amount and type of the feed sugars. In someembodiments, Km is from about 0.1 to about 100, from about 0.1 to about90, from about 0.1 to about 80, from about 0.1 to about 70, from about0.1 to about 60, from about 0.1 to about 50, from about 0.1 to about 40,from about 0.1 to about 30, from about 0.1 to about 25, from about 0.1to about 20, or from about 0.1 to about 15. In some embodiments, Km isfrom about 1 to about 100, from about 1 to about 90, from about 1 toabout 80, from about 1 to about 70, from about 1 to about 60, from about1 to about 50, from about 1 to about 40, from about 1 to about 30, fromabout 1 to about 25, from about 1 to about 20, from about 1 to about 15,from about 1 to about 10, from about 5 to about 50, from about 5 toabout 40, from about 5 to about 30, from about 5 to about 20, from about5 to about 15, or from about 5 to about 10. In some specificembodiments, Km is from about 1 to about 15 or from about 5 to about 15.

In some embodiments, an average, a standard deviation, and/or a relativestandard deviation are determined for the series of Km calculated. Asused herein, a relative standard deviation is expressed in percentage,and is obtained by multiplying the standard deviation by 100 anddividing this product by the average.

In some embodiments, the equilibrium is determined by the relativestandard deviation of the series of Km of less than 30%, less than 25%,less than 20%, less than 15%, less than 10%, less than 9%, less than 8%,less than 7%, less than 6%, less than 5%, less than 4%, less than 3%,less than 2%, or less than 1%. In some embodiments, the equilibrium isdetermined by the relative standard deviation of the series of Km ofless than 15%, less than 10%, or less than 5%.

G. Post-Reaction Steps

In some embodiments, a herein described method of manufacturingoligosaccharide preparations further comprises one or more additionalprocessing steps after heating the aqueous composition at a temperatureand for a sufficient time. In some embodiments, the additionalprocessing steps comprise, for example, separation (such aschromatographic separation), dilution, concentration, drying,filtration, demineralization, extraction, decolorization, or anycombination thereof. For example, in some embodiments, the methodcomprises a dilution step and a decolorization step. In someembodiments, the method comprises a filtration step and a drying step.

In some embodiments, the method comprises a dilution step, where wateris added into the oligosaccharide preparation to make a syrup ofoligosaccharide preparation. In some embodiments, the concentration ofoligosaccharide preparation in the syrup is from about 5% to about 80%,from about 10% to about 70%, from about 10% to about 60%, from about 10%to about 50%, from about 10% to about 40%, from about 10% to about 30%,or from about 15% to about 25%. In other embodiments, the method doesnot comprise a dilution step, but rather, the oligosaccharidepreparation is allowed to solidify. In some embodiments, the methodcomprises a filtration step. In some embodiments, the method comprisesrecycling the catalyst by filtration.

In some embodiments, the method described herein further comprises adecolorization step. In some embodiments, the oligosaccharidepreparation may undergo a decolorization step using any method known inthe art, including, for example, treatment with an absorbent, activatedcarbon, chromatography (e.g., using ion exchange resin), hydrogenation,and/or filtration (e.g., microfiltration).

In some embodiments, the oligosaccharide preparation is contacted with amaterial to remove salts, minerals, and/or other ionic species. Incertain embodiments, the oligosaccharide preparation is flowed throughan anionic/cationic exchange column pair. In one embodiment, the anionicexchange column contains a weak base exchange resin in a hydroxide formand the cationic exchange column contains a strong acid exchange resinin a protonated form.

In some embodiments, the method comprises a concentration step. In someembodiments, the centration step produces an oligosaccharide preparationwith increased concentration. For example, in some embodiments, theconcentration step comprises evaporation (e.g., vacuum evaporation),drying (e.g., freeze-drying and spray drying) or any combinationthereof.

In some embodiments, the method comprises an isolation step, wherein atleast a portion of the oligosaccharide preparation is separated. In someembodiments, the isolation step comprises crystallization,precipitation, filtration (e.g., vacuum filtration), and centrifugation,or any combination thereof.

In some embodiments, the method comprises a separation step. In someembodiments, the separation step comprises separating at least a portionof the oligosaccharide preparation from at least a portion of thecatalyst, from at least a portion of the unreacted feed sugars, or fromboth. In some embodiments, the separation step comprises filtration,chromatography, differential solubility, precipitation, extraction, orcentrifugation.

H Reactors

The methods described herein can comprise the use of one or morereactors suitable for sugar condensation, considering the reactiontemperature, pH, pressure, and other factors. In some embodiments, theone or more suitable reactors comprise a fed-batch stirred reactor, abatch stirred reactor, a continuous flow stirred reactor, a continuousplug-flow column reactor, an attrition reactor, or a reactor withstirring induced by an electromagnetic field. In some embodiments, theone or more suitable reactors comprise a reactor described in Ryu, S.K., and Lee, J. M., Bioconversion of waste cellulose by using anattrition bioreactor, Biotechnol. Bioeng. 25: 53-65 (1983); Gusakov, A.V., and Sinitsyn, A. P., Kinetics of the enzymatic hydrolysis ofcellulose: 1. A mathematical model for a batch reactor process, Enz.Microb. TechnoL, 7: 346-352 (1985); Gusakov, A. V., Sinitsyn, A. P.,Davydkin, I. Y., Davydkin, V. Y., Protas, O. V., Enhancement ofenzymatic cellulose hydrolysis using a novel type of bioreactor withintensive stirring induced by electromagnetic field, Appl. Biochem.Biotechnol., 56: 141-153 (1996); or Fernanda de Castilhos Corazza,Flavio Faria de Moraes, Gisella Maria Zanin and Ivo Neitzel, Optimalcontrol in fed-batch reactor for the cellobiose hydrolysis, ActaScientiarum. Technology, 25: 33-38 (2003).

In some embodiments, the one or more suitable reactors comprisefluidized bed, upflow blanket, immobilized, or extruder type reactorsfor hydrolysis and/or fermentation. In some embodiments, the one or moresuitable reactors comprise an open reactor, a closed reactor, or both.In some embodiments, where the method comprises a continuous process,the one or more suitable reactors can include a continuous mixer such asa screw mixer.

Process

In some embodiments, a herein described method of manufacturingoligosaccharide preparations comprises a batch process, a continuousprocess, or both. In some embodiments, the method of manufacturing theoligosaccharide preparation comprises a batch process. For example, insome embodiments of the batch process, manufacturing of subsequentbatches of the oligosaccharide preparation does not start until thecompletion of the current batch. In some embodiments, during the batchprocess, all or a substantial amount of oligosaccharide preparation isremoved from the reactor. In some embodiments, during the batch process,all the feed sugars and the catalyst are combined in a reactor beforethe aqueous composition is heated to the described temperature or beforethe polymerization is induced. In some embodiments, during the batchprocess, the feed sugars are added before, after, or simultaneous withthe addition of the catalyst.

In some embodiments, the batch process is a fed-batch process, whereinall the feed sugars are not added into the reactor at the same time. Insome embodiments of the fed-batch process, at least a portion of thefeed sugars are added into the reactor during polymerization or afterthe aqueous composition is heated to the described temperature. In someembodiments of the fed-batch process, at least 10%, 20%, 30%, 40%, 50%,or 60% by weight of the feed sugars are added into the reactor duringpolymerization or after the aqueous composition is heated to thedescribed temperature.

In some embodiments, the method of manufacturing the oligosaccharidepreparation comprises a continuous process. For example, in someembodiments of the continuous process, the contents of the reactorcontinuously flow through the reactor. In some embodiments, thecombination of the feed sugars with the catalyst and the removal of atleast a portion of the oligosaccharide preparation are performedconcurrently.

In some embodiments, the method of manufacturing the oligosaccharidepreparation comprises a single-pot or multi-pot process. For example, insome embodiments of the single-pot process, the polymerization isperformed in a single reactor. For another example, in some embodimentsof the multi-pot process, the polymerization is performed in more thanone reactor. In some embodiments of the multi-pot process, the methodcomprises 2, 3, or more reactors. In some embodiments of the multi-potprocess, the method comprises a combination step, where thepolymerization products from two or more reactors are combined.

I. Process

In some embodiments, the method of manufacturing the oligosaccharidepreparation comprises a batch process, a continuous process, or both. Insome embodiments, the method of manufacturing the oligosaccharidepreparation comprises a batch process. For example, in some embodimentsof the batch process, manufacturing of subsequent batches of theoligosaccharide preparation does not start until the completion of thecurrent batch. In some embodiments, during the batch process, all or asubstantial amount of oligosaccharide preparation is removed from thereactor. In some embodiments, during the batch process, all the feedsugars and the catalyst are combined in a reactor before the aqueouscomposition is heated to the described temperature or before thepolymerization is induced. In some embodiments, during the batchprocess, the feed sugars are added before, after, or simultaneous withthe addition of the catalyst.

In some embodiments, the batch process is a fed-batch process, whereinall the feed sugars are not added into the reactor at the same time. Insome embodiments of the fed-batch process, at least a portion of thefeed sugars are added into the reactor during polymerization or afterthe aqueous composition is heated to the described temperature. In someembodiments of the fed-batch process, at least 10%, 20%, 30%, 40%, 50%,or 60% by weight of the feed sugars are added into the reactor duringpolymerization or after the aqueous composition is heated to thedescribed temperature.

In some embodiments, the method of manufacturing the oligosaccharidepreparation comprises a continuous process. For example, in someembodiments of the continuous process, the contents of the reactorcontinuously flow through the reactor. In some embodiments, thecombination of the feed sugars with the catalyst and the removal of atleast a portion of the oligosaccharide preparation are performedconcurrently.

In some embodiments, the method of manufacturing the oligosaccharidepreparation comprises a single-pot or multi-pot process. For example, insome embodiments of the single-pot process, the polymerization isperformed in a single reactor. For another example, in some embodimentsof the multi-pot process, the polymerization is performed in more thanone reactor. In some embodiments of the multi-pot process, the methodcomprises 2, 3, or more reactors. In some embodiments of the multi-potprocess, the method comprises a combination step, where thepolymerization products from two or more reactors are combined.

IV. Nutritional Compositions Comprising Oligosaccharide Preparations

Provided herein are nutritional compositions comprising anoligosaccharide preparation. In certain embodiments, provided herein arenutritional compositions comprising a described oligosaccharidepreparation, wherein the presence and/or concentration of theoligosaccharide preparation within the nutritional compositions may beselectively determined and/or detected. Oligosaccharide preparations,which exhibit complex functional modulation of a microbial community,may be important components of nutritional compositions. Thus, thepresence and/or concentration of an oligosaccharide preparation withinnutritional compositions may be one of the factors that need to bemeasured in the quality control and manufacturing process of thenutritional compositions. Accordingly, the provided nutritionalcompositions are advantageous in terms of quality control andmanufacturing purposes as the presence and/or concentration of theoligosaccharide preparation may be selectively determined and/ordetected. For example, in some embodiments, the presence andconcentration of the oligosaccharide preparation may be determinedand/or detected by measuring a signal associated with theanhydro-subunit containing oligosaccharides.

In some embodiments, the nutritional composition is an animal feedcomposition. In some embodiments, the nutritional composition comprisesa base nutritional composition.

A. Base Nutritional Compositions

In some embodiments, the base nutritional composition comprises acarbohydrate source that is different from the oligosaccharidepreparation. For example, in some embodiments, the base nutritionalcomposition comprises a naturally occurring carbohydrate source such asstarch and plant fibers. In some embodiments, the base nutritionalcomposition comprises starch. In some embodiments, the base nutritionalcomposition comprises plant fibers.

In some embodiments, the base nutritional composition comprises one ormore carbohydrate sources that are derived from: seeds, roots, tubers,corn, tapioca, arrowroot, wheat, rice, potatoes, sweet potato, sago,beans (e.g., favas, lentils, mung beans, peas, and chickpeas.), maize,cassava, or other starchy foods (e.g., acorns, arrowroot, arracacha,bananas, barley, breadfruit, buckwheat, canna, colacasia, katakuri,kudzu, malanga, millet, oats, oca, polynesian arrowroot, sorghum, rye,taro, chestnuts, water chestnuts, and yams).

In some embodiments, the base nutritional composition comprises one ormore carbohydrate sources that are derived from: legumes (e.g., peas,soybeans, lupins, green beans, and other beans), oats, rye, chia,barley, fruits (e.g., figs, avocados, plums, prunes, berries, bananas,apple skin, quinces, and pears), vegetables (e.g., broccoli, carrots,cauliflower, zucchini, celery, nopal, and Jerusalem artichokes), roottubers, root vegetables (e.g., sweet potatoes and onions), psyllium seedhusks, seeds (e.g., flax seeds), nuts (e.g., almonds), whole grainfoods, wheat, corn bran, lignans, or any combination thereof. In someembodiments, the base nutritional composition comprises one or moreplant fibers derived from wheat bran, sugar beet pulp, fuzzycottonseeds, soy hulls, or any combination thereof.

In some embodiments, the base nutritional composition comprises lessthan 500 ppm, less than 400 ppm, less than 300 ppm, less than 200 ppm,less than 100 ppm, less than 50 ppm, less than 10 ppm, less than 5 ppm,or less than 1 ppm anhydro-subunits or anhydro-subunit containingoligosaccharides. In some embodiments, the base nutritional compositioncomprises less than 50 ppm, less than 10 ppm, less than 5 ppm, or lessthan 1 ppm anhydro-subunits or anhydro-subunit containingoligosaccharides. In some embodiments, the base nutritional compositionis essentially free of anhydro-subunits.

In some embodiments, the base nutritional composition lacks a detectablelevel of anhydro-subunits. Depending on the methods of detecting ordetermination, an anhydro-subunit level below a certain threshold can beundetectable. For example, in some embodiments, a detectable level ofanhydro-subunit can refer to at least 1000 ppm, at least 500 ppm, atleast 400 ppm, at least 300 ppm, at least 200 ppm, at least 100 ppm, atleast 50 ppm, at least 10 ppm, at least 5 ppm, or at least 1 ppm ofanhydro-subunit or anhydro-subunit containing oligosaccharides in thebase nutritional composition.

In some embodiments, the base nutritional composition comprises aplurality of oligosaccharides. In some embodiments, the base nutritionalcomposition comprises a glycosidic bond type distribution that isdifferent from the oligosaccharide preparation. For example, in someembodiments, the base nutritional composition comprises a higherpercentage of α-(1,4) glycosidic linkages than the oligosaccharidepreparation. In some embodiments, the glycosidic linkages such as theα-(1,4) glycosidic linkages in the base nutritional compositions aredigestible by one or more enzymes. In some embodiments, the glycosidiclinkages in the base nutritional composition are more readily digestibleand/or hydrolysable than the glycosidic linkages in the oligosaccharidepreparation.

In some embodiments, the level of α-(1,2) glycosidic linkage, α-(1,3)glycosidic linkage, α-(1,6) glycosidic linkage, β-(1,2) glycosidiclinkage, β-(1,3) glycosidic linkage, β-(1,4) glycosidic linkage, orβ-(1,6) glycosidic linkage in the base nutritional composition is atleast 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%,at least 13%, at least 14%, or at least 15% lower than the level of therespective glycosidic linkage in the oligosaccharide preparation. Insome embodiments, the level of α-(1,2) glycosidic linkage, α-(1,3)glycosidic linkage, α-(1,6) glycosidic linkage, β-(1,2) glycosidiclinkage, β-(1,3) glycosidic linkage, β-(1,4) glycosidic linkage, orβ-(1,6) glycosidic linkage in the base nutritional composition is atleast 10% lower than the level of the respective glycosidic linkage inthe oligosaccharide preparation.

In some embodiments, the level of α-(1,4) glycosidic linkage in the basenutritional composition is at least 50%, at least 40%, at least 35%, atleast 30%, at least 25%, at least 20%, at least 15%, at least 10%, atleast 5%, or at least 2% higher than the level of α-(1,4) glycosidiclinkage in the oligosaccharide preparation. In some embodiments, thelevel of α-(1,4) glycosidic linkage in the base nutritional compositionis at least 10% higher than the level of α-(1,4) glycosidic linkage inthe oligosaccharide preparation.

B. Animal Feed Composition

Depending on the type and age of an animal, a nutritional compositioncan comprise the oligosaccharide preparation and the base nutritionalcomposition at different ratio. For example, the oligosaccharidepreparation may be combined with the base nutritional composition atvarious ratios suitable for the type and age of an animal. In someembodiments, the oligosaccharide preparation is present in thenutritional composition at a concentration of from about 1 to about10000 ppm, from about 1 to about 5000 ppm, from about 1 to about 3000ppm, from about 1 to about 2000 ppm, from about 1 to about 1500 ppm,from about 1 to about 1000 ppm, from about 1 to about 500 ppm, fromabout 1 to about 250 ppm, from about 1 to about 100 ppm, from about 10to about 5000 ppm, from about 10 to about 3000 ppm, from about 10 toabout 2000 ppm, from about 10 to about 1500 ppm, from about 10 to about1000 ppm, from about 10 to about 500 ppm, from about 10 to about 250ppm, from about 10 to about 100 ppm, from about 50 to about 5000 ppm,from about 50 to about 3000 ppm, from about 50 to about 2000 ppm, fromabout 50 to about 1500 ppm, from about 50 to about 1000 ppm, from about50 to about 500 ppm, from about 50 to about 250 ppm, from about 50 toabout 100 ppm, from about 100 to about 5000 ppm, from about 100 to about3000 ppm, from about 100 to about 2000 ppm, from about 100 to about 1500ppm, from about 100 to about 1000 ppm, from about 100 to about 500 ppm,from about 100 to about 400 ppm, from about 100 to about 300 ppm, fromabout 100 to about 200 ppm, from about 200 to about 5000 ppm, from about200 to about 3000 ppm, from about 200 to about 2500 ppm, from about 200to about 2000 ppm, from about 200 to about 1500 ppm, from about 200 toabout 1000 ppm, from about 200 to about 500 ppm, from about 500 to about5000 ppm, from about 500 to about 3000 ppm, from about 500 to about 2500ppm, from about 500 to about 2000 ppm, from about 500 to about 1500 ppm,or from about 500 to about 1000 ppm. In some embodiments, theoligosaccharide preparation is present in the nutritional composition ata concentration of from about 1 to about 5000 ppm, from about 1 to about1000 ppm, from about 1 to about 500 ppm, from about 10 to about 5000ppm, from about 10 to about 2000 ppm, from about 10 to about 1000 ppm,from about 10 to about 500 ppm, from about 10 to about 250 ppm, fromabout 10 to about 100 ppm, from about 50 to about 5000 ppm, from about50 to about 2000 ppm, from about 50 to about 1000 ppm, from about 50 toabout 500 ppm, from about 50 to about 250 ppm, or from about 50 to about100 ppm. In some embodiments, the oligosaccharide preparation is presentin the nutritional composition at a concentration of from about 1 toabout 5000 ppm, from about 10 to about 1000 ppm, from about 10 to about500 ppm, or from about 50 to about 500 ppm.

In some embodiments, the oligosaccharide preparation is present in thenutritional composition at a concentration of greater than 10 ppm,greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, greaterthan 300 ppm, greater than 400 ppm, greater than 500 ppm, greater than600 ppm, greater than 1000 ppm, or greater than 2000 ppm. In someembodiments, the oligosaccharide preparation is present in thenutritional composition at a concentration of greater than 10 ppm,greater than 50 ppm, greater than 100 ppm, greater than 200 ppm, orgreater than 500 ppm.

In some embodiments, depending on the type and age of an animal, thenutritional composition can further comprise proteins, minerals (such ascopper, calcium, and zinc), salts, essential amino acids, vitamins,and/or antibiotics.

Also provided herein is a method of administering a nutritionalcomposition comprising a base nutritional composition and the disclosedoligosaccharide preparation to an animal. In some embodiments, theanimal is selected from cattle (e.g., beef cattle and dairy cattle),swine, aquatic animal, and poultry. In some embodiments, the animal isswine, such as sows, piglets, and hogs. In other embodiments, the animalis poultry such as chicken, duck, turkey, goose, quail, and hen. Inembodiments, the poultry is a broiler, a breeder, or a layer. In someembodiments, the animal is an aquatic animal such salmon, catfish, bass,eel, tilapia, flounder, shrimp, and crab. In some embodiments, thenutritional composition is administered to an animal in a dry form, aliquid form, a paste, or a combination thereof. In some embodiments, theform of administration, the feeding rate, and the feeding schedule canvary depending on the type and age of the animal.

C. Methods of Producing Nutritional Compositions

Provided herein are methods of manufacturing a nutritional compositioncomprising: combining an oligosaccharide preparation with a basenutritional composition. In some embodiments, the oligosaccharidepreparation comprises anhydro-subunit containing oligosaccharides. Insome embodiments, the oligosaccharide preparation comprises a glycosidicbond type distribution that is different from that of the basenutritional composition.

In some embodiments, the oligosaccharide preparation is a syntheticoligosaccharide preparation. In some embodiments, the syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions). In some embodiments, n isan integer greater than or equal to 2. In some embodiments, n is aninteger greater than 2. In some embodiments, n is an integer greaterthan or equal to 3. In some embodiments, n is an integer within a rangeof 1 to 100, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, or 50. In someembodiments, each of the DP1 to DPn fraction comprises from 0.1% to 90%anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry. In some embodiments, the DP1 and DP2fractions of the oligosaccharide preparation each independentlycomprises from about 0.1% to about 15% or from about 0.5% to about 10%of anhydro-subunit containing oligosaccharides by relative abundance asmeasured by mass spectrometry. In some embodiments, the DP1 and DP2fractions of the oligosaccharide preparation each independentlycomprises anhydro-subunit containing oligosaccharides within a range offrom about 0.1%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%,1.4%, or 1.5% to about 8%, 9%, 10%, 11%, 12%, 15% or 20% by relativeabundance as measured by mass spectrometry. In some embodiments, therelative abundance of oligosaccharides in each of the n fractionsdecreases monotonically with its degree of polymerization. In someembodiments, the relative abundance of oligosaccharides in at least 5,10, 20, or 30 DP fractions decreases monotonically with its degree ofpolymerization.

In some embodiments, the method of manufacturing a nutritionalcomposition comprises mixing the oligosaccharide preparation with thebase nutritional composition. For example, in some embodiments, themixing may be performed by an industrial blender and/or mixer such asdrum blender, double cone blender, ribbon blender, V blender, shearmixer, and paddle mixer.

In some embodiments, the method of manufacturing a nutritionalcomposition further comprises a herein described quality control step.In some embodiments, the herein described quality control step comprisesdetermining a level of a signal in a sample of the nutritionalcomposition and calculating a concentration of the oligosaccharidepreparation in the nutritional composition based on the level of thesignal. In some embodiments, the herein described quality control stepcomprises detecting a signal in a sample of the nutritional compositionthrough analytical instrumentation, and accepting or rejecting a batchof the nutritional composition based on the presence or absence of thesignal. In some embodiments, the herein described quality control stepcomprises detecting, through analytical instrumentation, the presence orabsence of a first signal in a first sample of the nutritionalcomposition, and a second signal in a second sample of the nutritionalcomposition, and comparing the first signal and the second signal. Insome embodiments, the signal, the first signal, and/or the second signalis/are (i) indicative of one or more anhydro-subunit containingoligosaccharides, (ii) associated with a degree of polymerization (DP)distribution of oligosaccharides, or (iii) associated with α-(1,2)glycosidic linkage, α-(1,3) glycosidic linkage, α-(1,6) glycosidiclinkage, β-(1,2) glycosidic linkage, β-(1,3) glycosidic linkage, β-(1,4)glycosidic linkage, or β-(1,6) glycosidic linkage of oligosaccharides.

Additionally, in some embodiments, the method of manufacturing anutritional composition comprises, after performing the quality controlstep, further mixing the oligosaccharide preparation with the basenutritional composition, adjusting the level of the oligosaccharidepreparation, or a combination thereof. In some embodiments, adjustingthe level of the oligosaccharide preparation comprises adding additionaloligosaccharide preparation into the nutritional composition or removinga portion of the oligosaccharide preparation from the nutritionalcomposition. In some embodiments, adjusting the level of theoligosaccharide preparation comprises adding additional base nutritionalcomposition into the nutritional composition or removing a portion ofthe base nutritional composition from the nutritional composition. Insome particular embodiments, adjusting the level of the oligosaccharidepreparation comprises adding additional oligosaccharide preparation intothe nutritional composition.

D. Animal Feed Pre-Mix

In some embodiments, the nutritional composition comprises an animalfeed pre-mix comprising a described oligosaccharide preparation.

In some embodiments, the animal feed pre-mix comprises a carriermaterial, which may be combined with the oligosaccharide preparation toproduce the animal feed pre-mix. In some embodiments, the carriermaterial may be any material in dry or liquid form that is suitable tobe combined with the oligosaccharide preparation in the nutritionalcomposition. In some embodiments, the carrier material comprises drieddistiller's grains, clay, vermiculite, diatamacious earth, hulls such asground rice hulls and ground oat hulls, silica such as feed grade silicagel and feed grade fumed silica, corn such as corn gluten feed, corngluten meal, and milled corn, or any combinations thereof. In someembodiments, the carrier material is milled corn. In other embodiments,the carrier material is ground rice hulls or ground oat hulls.

In some embodiments, the animal feed pre-mix is produced by combining acarrier material with the oligosaccharide preparation, both in a dryform. In some embodiments, the animal feed pre-mix is produced bycombining a carrier material with the oligosaccharide preparation; oneof the two is in a dry form. In some embodiments, the animal feedpre-mix is produced by combining a carrier material with theoligosaccharide preparation, both in a liquid form. For example, in someembodiments, an oligosaccharide preparation in a liquid form refers tothe oligosaccharide in a solution, e.g., an aqueous solution of theoligosaccharides such as syrup.

In some embodiments, the animal feed pre-mix is produced by combining acarrier material with a syrup comprising the oligosaccharidepreparation. In some embodiments, the concentration of theoligosaccharide preparation in the syrup is at least 40%, at least 45%,at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, or at least 80% by weight. In some embodiments, theconcentration of the oligosaccharide preparation in the syrup is aboutfrom 40% to 80%, 50% to 75%, or 60% to 70% by weight.

In some embodiments, the animal feed pre-mix is in a powder (e.g.,flowable powder), a slush, a slurry, a pellets form, or a liquid form.In some embodiments, the animal feed pre-mix has a moisture content ofless than 40%, 30%, 20%, 15%, 10%, or 5% by weight. In some embodiments,the animal feed pre-mix has a moisture content of less than 10% or 5% byweight. In some embodiments, the animal feed pre-mix has a moisturecontent of higher than 5%, 10%, 15%, 20%, 25%, or 30% by weight. Infurther embodiments, the moisture content of the animal feed pre-mix isadjusted to any of the described ranges. For example, in someembodiments, the animal feed pre-mix is dried to increase its moisturecontent to a described range.

In some embodiments, depending on a specific application, the animalfeed pre-mix comprises various levels of the oligosaccharidepreparation. In some embodiments, the animal feed pre-mix comprises atleast 1%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,95%, or 99% the oligosaccharide preparation by dry weight. In someembodiments, the animal feed pre-mix comprises at most 50%, 60%, 70%,80%, 90%, 95%, or 99% the oligosaccharide preparation by dry weight.

In some embodiments, the animal feed pre-mix or the carrier materialfurther comprises other animal nutrition such as minerals, fats, andproteins. In some embodiments, the carrier material or the animal feedpre-mix comprises copper, zinc, or both. In some embodiments, thecarrier material or the animal feed pre-mix comprises an ionophore orother coccidiostat. In some embodiments, the carrier material or theanimal feed pre-mix comprises an antibiotic. In some embodiments, thecarrier material comprises a carbohydrate source. In some embodiments,the carbohydrate source in the carrier material does not compriseanhydro-subunits. In some embodiments, the carbohydrate source in thecarrier material comprises a glycosidic bond type distribution that isdifferent from the glycosidic bond type distribution of theoligosaccharide preparation.

Accordingly, in some embodiments, the method of manufacturing anutritional composition comprises combining the animal feed pre-mix withthe base nutritional composition.

V. Methods of Providing Oligosaccharide Preparations to Animals

In some embodiments, the methods described herein include providingoligosaccharide preparations to animals. In certain variations, animalsare treated by being fed or provided an oligosaccharide preparation. Insome embodiments, the animals are provided an oligosaccharidepreparation at an intended specific dose. A specific dose may bequantified, for example, as the mass of oligosaccharide preparationconsumed by the animal per unit of time (e.g., grams per day), or as themass of oligosaccharide preparation consumed by the animal per unit oftime per unit animal body mass (e.g., mg of oligosaccharide per kg ofbody mass per day). In certain embodiments, the specific dose of anoligosaccharide preparation is 1, 5, 10, 15, 20, 25, 30, 40, 50, 60, 70,80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250,275, 300, 350, 400, 450, 500, 1000, 1500, 2000, 3000, 4000, 5000, or10000 mg/kg/day. In some embodiments, the mass of the oligosaccharidepreparation is measured as the DP1+ content on a dry solids basis. Insome embodiments, the mass of the oligosaccharide preparation ismeasured as the DP2+ content on a dry solids basis.

In some embodiments, animals are provided oligosaccharide preparationsby oral administration via nutritional compositions. In someembodiments, the nutritional composition is formulated to contain anoligosaccharide preparation at a fixed concentration or level ofinclusion. The oligosaccharide concentration or level of inclusion inthe nutritional composition can be quantified by, for example, the massfraction of the oligosaccharide preparation per total mass of the finalfeed or nutritional composition. In some embodiments, the concentrationor level of inclusion is measured in parts per million (ppm) ofoligosaccharide on a dry solids basis per final nutritional compositionon an as-is basis. In some embodiments, the concentration of theoligosaccharide preparation is measured as the mass fraction of DP1+species on a dry solids basis. In some embodiments, the concentration ofthe oligosaccharide preparation is measured as the mass fraction of DP2+species on a dry solids basis.

One of ordinary skill in the art would know various methods andtechniques for determining the concentration of the oligosaccharidepreparation in the nutritional composition or final feed to achieve anintended specific dose. For example, average daily feed intake as afunctional of age is established for different species of broilerchickens and might be used by a nutritionist or veterinarian todetermine a desired level of inclusion in final feed.

In some embodiments, animals are provided oligosaccharide preparationsby oral administration via consumed liquids. In some embodiments, theoligosaccharide preparations are provided via drinking water. In someembodiments, the concentration of the oligosaccharide preparation in thedrinking water is selected to provide an intended specific dose ofoligosaccharide preparation to the animal.

VI. Selectively Modulating Growth of Gastrointestinal Microbes A.Selectively Promoting the Growth of Beneficial Gastrointestinal Microbes

In some embodiments, the methods described herein include selectivelyenhancing or promoting the growth of one or more microbial (e.g.,bacterial) species in the gastrointestinal tract of an animal and insome embodiments quantifying the level of the one or more microbial(e.g., bacterial) species. In some embodiments, the make-up of thegastrointestinal microbiota of an animal is shifted by theoligosaccharide preparation toward that of a healthy state. In someembodiments, the microbial (e.g., bacterial) species is beneficial tothe animal (e.g., beneficial to the health).

In some embodiments, the microbial species is an archaea species. Insome embodiments, the microbial species is bacteria, virus,bacteriophage, parasite, fungi or protozoan species. In someembodiments, the microbial species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, organismsclassified as genera Bacteroides, Odoribacter, Oscillibacter,Subdoligranulum, Biophila, Barnesiella, or Ruminococcus. Exemplarybacteria also include, but are not limited to, organisms classified asgenera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium,or Streptococcus.

Microbial species include, but are not limited to, Bacteroides clarus,Bacteroides dorei, Odoribacter splanchinicus, and Barnesiellaintestinihominis.

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% of at least one microbial speciesclassified as genera Bacteroides, Odoribacter, Oscillibacter,Subdoligranulum, Biophila, Barnesiella, or Ruminococcus (e.g., asmeasured in a gastrointestinal sample as disclosed herein). In someembodiments, the animal has a gastrointestinal microbiota comprising atleast 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,35%, 40%, 45%, or 50% of at least one microbial species classified asgenera Enterococcus, Lactobacillus, Propionibacterium, Bifidobacterium,or Streptococcus (e.g., as measured in a gastrointestinal sample asdisclosed herein).

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45%, or 50% of at least one of Bacteroidesclarus, Bacteroides dorei, Odoribacter splanchinicus, or Barnesiellaintestinihominis (e.g., as measured in a gastrointestinal sample asdisclosed herein).

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 40%, or 50% of a combination of one or more microbialspecies classified as genera Bacteroides, Odoribacter, Oscillibacter,Subdoligranulum, Biophila, Barnesiella, or Ruminococcus (e.g., asmeasured in a gastrointestinal sample as disclosed herein). In someembodiments, the animal has a gastrointestinal microbiota comprising atleast 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%,40%, or 50% of a combination of one or more microbial species classifiedas genera Enterococcus, Lactobacillus, Propionibacterium,Bifidobacterium, or Streptococcus (e.g., as measured in agastrointestinal sample as disclosed herein).

In some embodiments, the animal has a gastrointestinal microbiotacomprising at least 0.1%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%,20%, 25%, 30%, 40%, or 50% of a combination of one or more ofBacteroides clarus, Bacteroides dorei, Odoribacter splanchinicus, orBarnesiella intestinihominis (e.g., as measured in a gastrointestinalsample as disclosed herein).

B. Selectively Inhibiting Growth of Pathogenic Gastrointestinal Microbes

In certain embodiments, the methods described herein include selectivelyreducing or inhibiting the growth of one or more microbial (e.g.,bacterial) species in the gastrointestinal tract of an animal and insome embodiments quantifying the level of the one or more microbial(e.g., bacterial) species. In some embodiments, the microbial (e.g.,bacterial) species is detrimental to the animal (e.g., detrimental tothe health of the animal). In some embodiments, the microbial (e.g.,bacterial) species is pathogenic to the animal. In some embodiments, themicrobial (e.g., bacterial) species is pathogenic to humans but not toanimals. In some embodiments, the make-up of the gastrointestinalmicrobiota of an animal is shifted by the oligosaccharide preparationtoward that of a healthy state.

In some embodiments, the microbial species is an archaea species. Insome embodiments, the microbial species is bacteria, virus,bacteriophage, parasite, fungi or protozoan species. In someembodiments, the microbial species is a bacterial species.

Bacteria disclosed herein include, but are not limited to, bacteria ofthe phylum Proteobacteria. Bacteria also include, but are not limitedto, organisms classified as genera Helicobacter, Escherichia,Salmonella, Vibrio, or Yersinia. Exemplary bacteria also include, butare not limited to, organisms classified as genera Treponema,Streptococcus, Staphylococcus, Shigella, Rickettsia, Orientia,Pseudomonas, Neisseria, Mycoplasma, Mycobacterium, Listeria, Leptospira,Legionella, Klebsiella, Haemophilus, Francisella, Ehrlichia,Enterococcus, Coxiella, Corynebacterium, Clostridium, Chlamydia,Chlamydophila, Campylobacter, Burkholderia, Brucella, Borrelia,Bordetella, Bifidobacterium, and Bacillus. Microbial species include,but are not limited to, Helicobacter pullorum, Proteobacteria johnsonii,Escherichia coli, Campylobacter jejuni, and Lactobacillus crispatus.Microbial species also include, but are not limited to, Aeromonashydrophila, Campylobacter fetus, Plesiomonas shigelloides, Bacilluscereus, Campylobacter jejuni, Clostridium botulinum, Clostridiumdifficile, Clostridium perfringens, enteroaggregative Escherichia coli,enterohemorrhagic Escherichia coli, enteroinvasive Escherichia coli,enterotoxigenic Escherichia coli, Helicobacter pylori, Klebsielliapneumonia, Lysteria monocytogenes, Plesiomonas shigelloides, Salmonellaspp., Salmonella typhi, Salmonella paratyphi, Shigella spp.,Staphylococcus spp., Staphylococcus aureus, vancomycin-resistantenterococcus spp., Vibrio spp., Vibrio cholerae, Vibrioparahaemolyticus, Vibrio vulnificus, and Yersinia enterocolitica. Insome embodiments, the bacterium is singe or multi drug resistant.

In some embodiments, the animal has a gastrointestinal microbiotacomprising less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1% or 0.1% bacteria classified as genera Helicobacter,Proteobacteria, Escherichia, Campylobacter, or Lactobacillus (e.g., asmeasured in a gastrointestinal sample as disclosed herein). In someembodiments, the combination of bacteria classified as generaHelicobacter, Proteobacteria, Escherichia, Campylobacter, orLactobacillus is less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%,5%, 4%, 3%, 2%, 1% or 0.1% of the gastrointestinal microbiota of theanimal (e.g., as measured in a gastrointestinal sample as disclosedherein).

In some embodiments, the animal has a gastrointestinal microbiotacomprising less than 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%,4%, 3%, 2%, 1% or 0.1% Helicobacter pullorum, Proteobacteria johnsonii,Escherichia coli, Campylobacter jejuni, or Lactobacillus crispatus(e.g., as measured in a gastrointestinal sample as disclosed herein). Insome embodiments, the combination of Helicobacter pullorum,Proteobacteria johnsonii, Escherichia coli, Campylobacter jejuni, orLactobacillus crispatus is less than 50%, 40%, 30%, 20%, 15%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or 0.1% of the gastrointestinalmicrobiota of the animal (e.g., as measured in a gastrointestinal sampleas disclosed herein).

C. Sampling and Detecting Gastrointestinal Microbes

In certain embodiments, the methods described herein include detectingor quantifying one or more microbial (e.g., bacterial) species in thegastrointestinal microbiota of an animal. In certain embodiments, themicrobial (e.g., bacterial) species is detected or quantified in agastrointestinal microbiota sample from an animal. Gastrointestinalmicrobiota samples can be obtained from an animal in any standard formwhich reflects the microbial contents of the gastrointestinal tract ofthe animal. Gastrointestinal microbiota samples include gastrointestinaltissue samples obtained e.g., by endoscopic biopsy. Gastrointestinaltissues include, e.g., oral tissue, esophagus, stomach, intestine,ileum, cecum, colon or rectum. Samples also feces, saliva, andgastrointestinal ascites. Methods of obtaining gastrointestinalmicrobiota samples are standard and known to the skilled artisan.

In some embodiments, the sample is a single sample from a single animal.In some embodiments, the sample is a combination of multiple samplesfrom a single animal. In some embodiments, microbes (e.g., bacteria(e.g., total bacteria)) are purified from the sample prior to analysis.In some embodiments, microbes (e.g., bacteria) from a single sample arepurified. In some embodiments, microbes (e.g., bacteria) from multiplesamples from a single animal are purified and subsequently combinedprior to analysis.

In some embodiments, total DNA or total RNA is isolated from the sample.Genomic DNA can be extracted from samples using standard methods knownto the skilled artisan and including commercially available kits, suchas the Mo Bio Powersoil®-htp 96 Well Soil DNA Isolation Kit (Mo BioLaboratories, Carlsbad, Calif.), the Mo Bio Powersoil® DNA Isolation Kit(Mo Bio Laboratories, Carlsbad, Calif.), or the QIAamp DNA Stool MiniKit (QIAGEN, Valencia, Calif.) according to the manufacturer'sinstructions. RNA can be extracted from samples using standard assaysknown to the skilled artisan including commercially available kits, suchas the RNeasy PowerMicrobiome Kit (QIAGEN, Valencia, Calif.) andRiboPure Microbial RNA Purification Kit (Life Technologies, Carlsbad,Calif.). Another method for isolation of microbial (e.g., bacterial) RNAmay involve enrichment of mRNA in purified samples of microbial RNAthrough removal of tRNA. Alternatively, RNA may be converted to cDNA,which can be used to generate sequencing libraries using standardmethods such as the Nextera XT Sample Preparation Kit (Illumina, SanDiego, Calif.).

Identification and determination of the relative abundance of amicrobial (bacterial) species in a sample may be determined by standardmolecular biology methods known to the skilled artisan, including e.g.,genetic analysis (e.g. DNA sequencing (e.g., full genome sequencing,whole genome shotgun sequencing (WSG)), RNA sequencing, PCR,quantitative PCR (qPCR)), serology and antigen analysis, microscopy,metabolite identification, gram staining, flow cytometry, immunologicaltechniques, and culture based methods such as counting colony formingunits.

In some embodiments, identification and relative abundance of amicrobial (e.g., bacterial) species is determined by whole genome shotgun sequencing (WGS), wherein extracted DNA is fragmented into pieces ofvarious lengths (from 300 to about 40,000 nucleotides) and directlysequenced without amplification. Sequence data can be generated usingany sequencing technology including for example, but not limited toSanger, Illumina, 454 Life Sciences, Ion Torrent, ABI, PacificBiosciences, and/or Oxford Nanopore.

Sequencing libraries for microbial whole-genome sequencing (WGS) may beprepared from microbial (e.g., bacterial) genomic DNA. For genomic DNAthat has been isolated from an animal sample, the DNA may optionally beenriched for microbial DNA using commercially available kits, forexample, the NEBNext Microbiome DNA Enrichment Kit (New England Biolabs,Ipswich, Mass.) or other enrichment kit. Sequencing libraries may beprepared from the genomic DNA using commercially available kits as well,such as the Nextera Mate-Pair Sample Preparation Kit, TruSeq DNAPCR-Free or TruSeq Nano DNA, or the Nextera XT Sample Preparation Kit(Illumina, San Diego, Calif.) according to the manufacturer'sinstructions.

Alternatively, libraries can be prepared using other kits compatiblewith the Illumina sequencing platform, such as the NEBNext DNA LibraryConstruction Kit (New England Biolabs, Ipswich, Mass.). Libraries maythen be sequenced using standard sequencing technology including, butnot limited to, a MiSeq, HiSeq or NextSeq sequencer (Illumina, SanDiego, Calif.).

Alternatively, a whole genome shotgun fragment library prepared usingstandard methods in the art may be used. For example, the shotgunfragment library could be constructed using the GS FLX Titanium RapidLibrary Preparation Kit (454 Life Sciences, Branford, Conn.), amplifiedusing a GS FLX Titanium emPCR Kit (454 Life Sciences, Branford, Conn.),and sequenced following standard 454 pyrosequencing protocols on a 454sequencer (454 Life Sciences, Branford, Conn.).

Nucleic acid sequences can be analyzed to define taxonomic assignmentsusing sequence similarity and phylogenetic placement methods or acombination of the two strategies. A similar approach can be used toannotate protein names, protein function, transcription factor names,and any other classification schema for nucleic acid sequences. Sequencesimilarity based methods include BLAST, BLASTx, tBLASTn, tBLASTx,RDP-classifier, DNAclust, RapSearch2, DIAMOND, USEARCH, and variousimplementations of these algorithms such as QIIME or Mothur. Thesemethods map a sequence read to a reference database and select the bestmatch. Common databases include KEGG, MetaCyc, NCBI non-redundantdatabase, Greengenes, RDP, and Silva for taxonomic assignments. Forfunctional assignments, reads are mapped to various functional databasessuch as COG, KEGG, BioCyc, MetaCyc, and the Carbohydrate-Active Enzymes(CAZy) database. Microbial clades are assigned using software includingMetaPhlAn.

In some embodiments, the microbial constituents are identified bycharacterizing the DNA sequence of microbial 16S small subunit ribosomalRNA gene (16S rRNA gene). 16S rRNA gene is approximately 1,500nucleotides in length, and in general is highly conserved acrossorganisms, but contain specific variable and hypervariable regions(V1-V9) that harbor sufficient nucleotide diversity to differentiatespecies- and strain-level taxa of most organisms. These regions inbacteria are defined by nucleotides 69-99, 137-242, 433-497, 576-682,822-879, 986-1043, 1117-1173, 1243-1294 and 1435-1465 respectively usingnumbering based on the E. coli system of nomenclature.

Composition of a microbial community can be deduced by sequencing full16S rRNA gene, or at least one of the VI, V2, V3, V4, V5, V6, V7, V8,and V9 regions of this gene or by sequencing of any combination ofvariable regions from this gene (e.g. V1-3 or V3-5). In one embodiment,the VI, V2, and V3 regions are used to characterize a microbiota. Inanother embodiment, the V3, V4, and V5 regions are used to characterizea microbiota. In another embodiment, the V4 region is used tocharacterize a microbiota.

Sequences that are at least 97% identical to each other are grouped intoOperational Taxonomic Units (OTUs). OTUs that contain sequences with 97%similarity correspond to approximately species level taxa. At least onerepresentative sequence from each OTU is chosen and is used to obtain ataxonomic assignment for an OTU by comparison to a reference database ofhighly curated 16S rRNA gene sequences (such as Greengenes or SILVAdatabases). Relationship between OTUs in a microbial community could bededuces by constructing a phylogenetic tree from representativesequences from each OTU. Using known techniques, in order to determinethe full 16S sequence or the sequence of any variable region of the 16Ssequence, genomic DNA is extracted from a microbial sample, the 16S rRNA(full region or specific variable regions) amplified using polymerasechain reaction (PCR), the PCR products are cleaned, and nucleotidesequences delineated to determine the genetic composition of 16S rRNAgene or a variable region of the gene. If full 16S sequencing isperformed, the sequencing method used may be, but is not limited to,Sanger sequencing. If one or more variable regions is used, such as theV4 region, the sequencing can be, but is not limited to being performedusing the Sanger method or using a next-generation sequencing method,such as an Illumina method. Primers designed to anneal to conservedregions of 16S rRNA genes (e.g., the 515F and 805R primers foramplification of the V4 region) could contain unique barcode sequencesto allow characterizing multiple microbial communities simultaneously.

In addition to the 16S rRNA gene, a selected set of genes that are knownto be marker genes for a given species or taxonomic group is analyzed toassess the composition of a microbial community. These genes arealternatively assayed using a PCR-based screening strategy. For example,various strains of pathogenic Escherichia coli are distinguished usinggenes that encode heat-labile (LTI, LTIIa, and LTIIb) and heat-stable(STI and STII) toxins, verotoxin types 1, 2, and 2e (VT1, VT2, and VT2e,respectively), cytotoxic necrotizing factors (CNF1 and CNF2), attachingand effacing mechanisms (eaeA), enteroaggregative mechanisms (Eagg), andenteroinvasive mechanisms (Einv). The optimal genes to utilize todetermine the taxonomic composition of a microbial community by use ofmarker genes are familiar to one with ordinary skill in the art ofsequence based taxonomic identification.

In some embodiments, the identity of the microbial composition ischaracterized by identifying nucleotide markers or genes, in particularhighly conserved genes (e.g., “house-keeping” genes), or a combinationthereof. Using defined methods, DNA extracted from a microbial samplewill have specific genomic regions amplified using PCR and sequenced todetermine the nucleotide sequence of the amplified products.

D. Microbial Gene Transcription

In certain embodiments, the methods described herein include promotingexpression of one or more microbial (e.g., bacterial) protein in thegastrointestinal tract of an animal. In some embodiments, the microbialprotein is a bacterial protein. In certain embodiments the microbial(e.g., bacterial) protein is a hydrolytic enzyme, a protein involved indigestion (e.g. hydrolytic enzymatic digestion), or a protein involvedin metabolism. Microbial proteins include, but are not limited to, acarbohydrate active enzyme (CAZymes), a sus-like polysaccharideutilization loci (PULs) protein, GH127 (CAZyme α-L-arabinofuranosidase),susC (outer membrane protein involved in starch binding) and susD(starch binding protein).

Microbial (e.g., bacterial) proteins described herein can be detectedand quantified using standard molecular biology techniques. For example,the level of expression on a microbial protein can be detected in agastrointestinal sample of an animal by RNA (e.g. RNA sequencing,shotgun sequencing, quantitative PCR) or protein expression (e.g. ELISA,western blot, other immunological techniques).

VII. Methods of Enhancing Animal Performance A. Feed Conversion Ratio

In some embodiments, the methods described herein include reducing thefeed conversion ratio of an animal. In some embodiments, an animaladministered a synthetic oligosaccharide preparation, a nutritionalcomposition, an animal feed pre-mix, or an animal feed composition asdescribed herein has a lower feed conversion ratio compared to an animalprovided a diet that does not include the synthetic oligosaccharidepreparation. As used herein the term “feed conversion ratio (FCR),”refers to the ratio of feed mass input (for example consumed by theanimal) to the animal output, wherein the animal output is the targetanimal product. For example, the animal output for dairy animals ismilk, whereas the animal output for animals raised for meat is bodymass.

In some embodiments, the animal is raised for meat, and the targetanimal output is body mass. Thus, in some embodiments, the FCR refers tothe ratio of the weight of feed consumed compared to the final bodyweight of the animal prior to processing. In some embodiments, the FCRrefers to the ratio of the weight of feed consumed compared to the finalbody weight gain of the animal prior to processing. It should beunderstood that FCR may be measured for an animal or population ofanimals over different time periods. For example, in some embodiments,the FCR is an FCR over the entire lifetime of the animal. In otherembodiments, the FCR is a daily FCR, or a weekly FCR, or a cumulativeFCR measured up until a particular moment in time (for example, aparticular day).

A person of skill in the art would recognize that the performance targetminimum FCR (optimal FCR) may be different for different types ofanimals and may be different for different breeds of one type animal(for example, different breeds of broiler chickens, or different breedsof swine). The performance target minimum FCR may also be differentdepending on age of the animal (for example, chickens or swine in agrower phase compared to a finisher phase), or the sex of the animal. Itshould be clear that the optimal FCR may be different depending on anycombination of these factors.

Performance target minimum generally refers to the lowest feedefficiency observed for a given animal and breed under ideal growingconditions, ideal animal health, and ideal dietary nutrition. It is wellknown to one skilled in the art that under common growing conditions, ananimal may not achieve the performance target minimum FCR. An animal maynot achieve its performance target minimum FCR due to a variety ofhealth, nutrition, environmental, and/or community influences. An animalmay not achieve its performance target minimum FCR when raised in achallenged environment, which may include, for example, environmentalpathogenic stress, excessive environmental temperature (heat stress),excessive environmental humidity, crowding, or other social interactioneffects, such as difficulty accessing feed or drinking water. In someembodiments, an animal may not achieve its performance target minimumFCR due to disease or environmental pathogenic stress. In otherembodiments, an animal may not achieve its performance target minimumFCR due to excessive environmental temperature (heat stress), orexcessive environmental humidity. In yet other embodiments, an animalmay not achieve its performance target minimum FCR due to crowding, orother social interaction effects, such as difficulty accessing feed ordrinking water.

In some embodiments, an animal provided a diet which does not includethe synthetic oligosaccharide preparation described herein has an FCRthat is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher thanthe performance target minimum FCR. In certain embodiments, an animalprovided a diet which does not include a synthetic oligosaccharidepreparation described herein has an FCR that is 1% to 10% higher thanthe performance target minimum, 2% to 10% higher than the performancetarget minimum, or 5% to 10% higher than the performance target minimum.

In some embodiments, an animal provided a nutritional compositioncomprising a synthetic oligosaccharide preparation, a nutritionalcomposition, an animal feed pre-mix, or an animal feed composition asdescribed herein has an FCR that is closer to the performance targetminimum compared to an animal provided a diet that does not include thesynthetic oligosaccharide preparation. In particular embodiments, theanimal provided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition asdescribed herein has an FCR that is between 0 to 10% higher than theperformance target minimum, between 0 to 5% higher than the performancetarget minimum, or between 0 to 2% higher than the performance targetminimum.

In some embodiments, an animal provided a synthetic oligosaccharidepreparation, a nutritional composition, animal feed pre-mix, or animalfeed composition as described herein has a lower feed conversion ratiocompared to an animal provided a diet that does not include thesynthetic oligosaccharide preparation. For example, in certainembodiments, the animal provided a diet comprising the syntheticoligosaccharide preparation consumes less food but has the same animaloutput as compared to an animal provided a diet that does not includethe synthetic oligosaccharide preparation. In other embodiments, theanimal provided a diet comprising the synthetic oligosaccharidepreparation consumes the same amount of food but has a higher animaloutput as compared to an animal provided a diet that does not includethe synthetic oligosaccharide preparation. In yet other embodiments, theanimal provided a diet comprising the synthetic oligosaccharidepreparation consumes less food and has a higher animal output ascompared to an animal provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the FCR of an animal provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition as described herein is reduced atleast 1%, at least 2%, at least 4%, at least 6%, at least 8%, at least10%, at least 12%, between 1 to 10%, between 4 to 10%, between 1 to 8%,between 4 to 8%, between 1 to 6%, or between 4 to 6% as compared to ananimal provided a diet that does not include the syntheticoligosaccharide preparation. In some embodiments, the animal is poultry.In certain embodiments, the FCR of the poultry is reduced over 0 to 14days of age, over 15 to 28 days of age, over 29 to 35 days of age, over35 days, over 42 days, over 6 weeks, over 6.5 weeks, over 0 to 35 daysof age, over 0 to 42 days of age, over 0 to 6 weeks of age, over 0 to6.5 weeks of age, over 15 to 35 days of age, over 36 to 42 days of age,over 15 to 39 days of age, or over 40 to 46 days of age.

In one embodiment, the FCR over 35 days for poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition as described herein is reduced bybetween 4 to 6% as compared to poultry provided a diet that does notinclude the synthetic oligosaccharide preparation. For example, in acertain embodiment, the FCR over 35 days for poultry provided anutritional composition describing a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition as described herein is 1.53, the FCR over 35 days forpoultry provided a diet without the synthetic oligosaccharidepreparation is 1.61, and the FCR of the poultry provided the nutritionalcomposition comprising a oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition is reducedabout 5% compared to the poultry provided a diet without the syntheticoligosaccharide preparation. In some embodiments, the FCR over 42 days,over 6 weeks, or over 6.5 weeks days for poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition as described herein is reduced bybetween 4 to 6% as compared to poultry provided a diet that does notinclude the synthetic oligosaccharide preparation.

In some embodiments, an animal population provided a syntheticoligosaccharide preparation, nutritional preparation, animal feedpre-mix, or animal feed composition as described herein has a lower FCRcompared to an animal population provided a diet that does not includethe synthetic oligosaccharide preparation, wherein the FCR is correctedfor mortality in the animal population.

In certain embodiments, an animal provided a synthetic oligosaccharidepreparation, animal feed pre-mix, or animal feed composition has a lowerFCR than an animal provided a diet that does not include the syntheticoligosaccharide preparation, but which does include one or moreantibiotics, one or more ionophores, soluble corn fiber, modified wheatstarch, or yeast mannan, or any combinations thereof.

It is known to one skilled in the art, that when determining FCR, theFCR may be adjusted for mortality to reduce noise due to small numberstatistics. Methods for adjusting FCR for mortality are well known toone skilled in the art.

In some embodiments that may be combined with any of the foregoingembodiments, the poultry is an individual poultry, while in otherembodiments the poultry is a poultry population.

In some embodiments, the animal is poultry, and the animal feedcomposition is poultry feed, wherein the synthetic oligosaccharidepreparation, poultry nutritional composition, poultry feed pre-mix, orpoultry feed composition feed reduces feed conversion ratio (FCR) by upto about 10%, or about 5%, or between 1% and 10%, between 2% and 10%,between 3% and 10%, between 4% and 10%, between 5% and 10%, between 2%and 5%, between 2% and 6%, between 2% and 7%, between 2% and 8%, between2% and 9%, or between 1% and 5%, when fed to poultry as compared topoultry fed a feed composition without the synthetic oligosaccharidepreparation.

In certain embodiments, the poultry suffers from a disease or adisorder, or is raised in a challenged environment, wherein thesynthetic oligosaccharide preparation, poultry nutritional composition,poultry feed pre-mix, or poultry feed composition feed reduces feedconversion ratio (FCR) by up to about 30%, about 25%, about 20%, about15%, about 10%, or about 5%, or between 1% and 30%, between 5% and 30%,between 10% and 30%, between 5% and 20%, between 10% and 20%, between 1%and 20%, between 1% and 15%, between 1% and 10%, between 2% and 10%,between 3% and 10%, between 4% and 10%, between 5% and 10%, between 2%and 5%, between 2% and 6%, between 2% and 7%, between 2% and 8%, between2% and 9%, or between 1% and 5%, when fed to poultry as compared topoultry fed a feed composition without the synthetic oligosaccharidepreparation.

In some embodiments, the animal is swine, and the animal feedcomposition is swine feed, wherein the synthetic oligosaccharidepreparation, swine nutritional composition, swine feed pre-mix, or swinefeed composition reduces feed conversion ratio (FCR) by up to about 15%,about 10%, or about 5%, or between 1% and 15%, between 2% and 15%,between 3% and 15%, between 4% and 15%, between 5% and 15%, between 10%and 15%, between 1% and 10%, between 2% and 10%, between 3% and 10%,between 4% and 10%, between 5% and 10%, between 2% and 5%, between 2%and 6%, between 2% and 7%, between 2% and 8%, between 2% and 9%, orbetween 1% and 5%, when fed to swine as compared to swine fed a feedcomposition without the synthetic oligosaccharide preparation.

In certain embodiments, the swine suffers from a disease or a disorder,or is raised in a challenged environment, wherein the syntheticoligosaccharide preparation, swine nutritional composition, swine feedpre-mix, or swine feed composition reduces feed conversion ratio (FCR)by up to about 40%, about 35% about 30%, about 25%, about 20%, about15%, about 10%, or about 5%, or between 1% and 40%, between 5% and 40%,between 10% and 40%, between 15% and 40%, between 20% and 40%, between25% and 40%, between 30% and 40%, between 1% and 30%, between 5% and30%, between 10% and 30%, between 5% and 20%, between 10% and 20%,between 1% and 20%, between 1% and 15%, between 1% and 10%, between 2%and 10%, between 3% and 10%, between 4% and 10%, between 5% and 10%,between 2% and 5%, between 2% and 6%, between 2% and 7%, between 2% and8%, between 2% and 9%, or between 1% and 5%, when fed to swine ascompared to swine fed a feed composition without the syntheticoligosaccharide preparation.

B. Body Weight

In some embodiments, a subject animal that is fed a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition described herein may experience anincrease in weight gain, compared to a control animal that is not fedthe oligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, both thesubject animal and the control animal consume the same quantity of feedon a weight basis, but the subject animal provided the syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition experiences an increase in weightgain compared to the control animal that is fed a diet that does notinclude the synthetic oligosaccharide preparation.

The weight gain of an animal may be determined by any suitable methodsknown in the art. For example, to determine weight gain of an animalthat is subjected to a feeding regimen of the synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition, one of skill in the art can measure the mass of ananimal prior to the feeding regimen, measure the mass of the animalafter the animal is fed the synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feedcomposition, and determine the difference between those twomeasurements.

In some embodiments, the weight gain may be an average daily weight gain(also referred to as average daily gain (ADG)), an average weekly weightgain (AWG), or a final body weight gain (BWG).

C. Average Daily Weight Gain

In some embodiments, providing an animal with a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition results in an increased averagedaily weight gain than an animal provided feed without the syntheticoligosaccharide preparation. In some embodiments, providing an animalpopulation with a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition results inan increased average daily weight gain than an animal populationprovided feed without the synthetic oligosaccharide preparation.

In one embodiment, the average daily weight gain for an animal is theweight gained each day by an individual animal, averaged over a givenperiod of time. In some embodiments, the average daily weight gain foran animal population is the average daily weight gain for eachindividual animal, averaged over the population; wherein the averagedaily weight gain is the weight gained each day by the individualanimal, averaged over a given period of time. In yet other embodiments,the average daily weight gain for an animal population is the totalweight gained by the population each day, divided by the number ofindividual animals in the population, averaged over a given period oftime. It should be understood that the daily weight gain or averagedaily weight gain may be further averaged, for example to provide anaverage daily weight gain across animal populations.

In certain embodiments, the animal is poultry, and the poultry provideda synthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has an average daily weightgain of at least 20 grams per day, at least 30 grams per day, at least40 grams per day, at least 50 grams per day, at least 60 grams per day,at least 70 grams per day, at least 80 grams per day, at least 90 gramsper day, between 20 to 100 grams per day, between 20 to 80 grams perday, between 30 to 50 grams per day, between 40 to 60 grams per day,between 50 to 70 grams per day, or between 70 to 90 grams per day. Inone embodiment, the animal is poultry, and the poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has an average daily weightgain of at least 50 grams per day. In certain embodiments, the poultryprovided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition has anaverage daily weight gain of at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 6%, at least 8%, at least 9%, at least10%, at least 11%, at least 12%, between 1 to 10%, between 2 to 8%, orbetween 3 to 5% greater than the average daily weight gain of poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In certain embodiments, the animal is poultry, and the poultry isbetween 0 to 14 days of age, and the average daily weight gain is atleast 30 grams, at least 40 grams, or at least 50 grams per day.

In other embodiments, the animal is poultry, the poultry is between 14to 28 days of age, and the average daily weight gain is at least 70grams, at least 80 grams, or at least 90 grams per day.

In still other embodiments, the animal is poultry, the poultry isbetween 29 to 35 days of age, and the average daily weight gain is atleast 50 grams, at least 60 grams, or at least 70 grams per day.

In some embodiments that may be combined with the foregoing, the animalis poultry, and the animal feed composition is poultry feed, wherein thesynthetic oligosaccharide preparation, poultry nutritional composition,poultry feed pre-mix, or poultry feed composition increases averagedaily gain in poultry by up to about 10%, or about 5%, or between 1% and10%, between 2% and 10%, between 3% and 10%, between 4% and 10%, between5% and 10%, between 2% and 5%, between 2% and 6%, between 2% and 7%,between 2% and 8%, between 2% and 9%, or between 1% and 5%, when fed tothe poultry as compared to poultry fed a feed composition without thesynthetic oligosaccharide preparation.

In certain embodiments, the poultry suffers from a disease or adisorder, or is raised in a challenged environment, wherein thesynthetic oligosaccharide preparation, poultry nutritional composition,poultry feed pre-mix, or poultry feed composition increases averagedaily gain in poultry by up to about 30%, about 25%, about 20%, about15%, about 10%, or about 5%, or between 1% and 30%, between 5% and 30%,between 10% and 30%, between 5% and 20%, between 10% and 20%, between 1%and 20%, between 1% and 15%, between 1% and 10%, between 2% and 10%,between 3% and 10%, between 4% and 10%, between 5% and 10%, between 2%and 5%, between 2% and 6%, between 2% and 7%, between 2% and 8%, between2% and 9%, or between 1% and 5%, when fed to the poultry as compared topoultry fed a feed composition without the synthetic oligosaccharidepreparation.

In some embodiments that may be combined with the foregoing, the animalis swine, and the animal feed composition is swine feed, wherein thesynthetic oligosaccharide preparation, swine nutritional preparation,swine feed pre-mix, or swine feed composition increases average dailygain in swine by up to about 15%, about 10%, or about 5%, or between 1%and 15%, between 2% and 15%, between 3% and 15%, between 4% and 15%,between 5% and 15%, between 10% and 15%, between 1% and 10%, between 2%and 10%, between 3% and 10%, between 4% and 10%, between 5% and 10%,between 2% and 5%, between 2% and 6%, between 2% and 7%, between 2% and8%, between 2% and 9%, or between 1% and 5%, when fed to swine ascompared to swine fed a feed composition without the syntheticoligosaccharide preparation.

In certain embodiments, the swine suffers from a disease or a disorder,or is raised in a challenged environment, wherein the oligosaccharidepreparation, swine nutritional composition, swine feed pre-mix, or swinefeed composition increases average daily gain in swine by up to about40%, about 35% about 30%, about 25%, about 20%, about 15%, about 10%, orabout 5%, or between 1% and 40%, between 5% and 40%, between 10% and40%, between 15% and 40%, between 20% and 40%, between 25% and 40%,between 30% and 40%, between 1% and 30%, between 5% and 30%, between 10%and 30%, between 5% and 20%, between 10% and 20%, between 1% and 20%,between 1% and 15%, between 1% and 10%, between 2% and 10%, between 3%and 10%, between 4% and 10%, between 5% and 10%, between 2% and 5%,between 2% and 6%, between 2% and 7%, between 2% and 8%, between 2% and9%, or between 1% and 5%, when fed to swine as compared to swine fed afeed composition without the synthetic oligosaccharide preparation.

In certain embodiments, the animal is swine, and the swine provided asynthetic oligosaccharide preparation, swine nutritional preparation,swine feed pre-mix, or swine feed composition has an average dailyweight gain of at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3 to 5%greater than the average daily weight gain of swine provided a diet thatdoes not include the synthetic oligosaccharide preparation.

D. Average Weekly Weight Gain

In some embodiments, providing an animal with a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition results in an increased averageweekly weight gain than an animal provided feed without the syntheticoligosaccharide preparation. In some embodiments, providing an animalpopulation with a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition results inan increased average weekly weight gain than an animal populationprovided feed without the synthetic oligosaccharide preparation.

In one embodiment, the average weekly weight gain for an animal is theweight gained each week by an individual animal, averaged over a givenperiod of time. In some embodiments, the average weekly weight gain foran animal population is the average weekly weight gain for eachindividual animal, averaged over the population; wherein the averageweekly weight gain is the weight gained each week by the individualanimal, averaged over a given period of time. In yet other embodiments,the average weekly weight gain for an animal population is the totalweight gained by the population each week, divided by the number ofindividual animals in the population, averaged over a given period oftime. It should be understood that the average weekly weight gain may befurther averaged, for example to provide an average weekly weight gainacross animal populations.

In certain embodiments, the animal is poultry, and poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has an average weekly weightgain of at least 100 grams per week, at least 200 grams per week, atleast 300 grams per week, at least 400 grams per week, at least 500grams per week, at least 600 grams per week, at least 700 grams perweek, at least 800 grams per week, between 100 to 800 grams per week,between 100 to 400 grams per week, between 300 to 600 grams per week,between 500 to 800 grams per week, or between 350 to 550 grams per week.In one embodiment, poultry provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition has an average weekly weight gain of at least 400 gramsper week. In certain embodiments, poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition has an average weekly weight gain ofat least 1%, at least 2%, at least 3%, at least 4%, at least 5%, atleast 6%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, between 1 to 10%, between 2 to 8%, or between 3 to 5% greater thanthe average weekly weight gain of poultry provided a diet that does notinclude the oligosaccharide preparation.

In certain embodiments, the animal is swine, and swine provided asynthetic oligosaccharide preparation, swine nutritional composition,swine feed pre-mix, or swine feed composition has an average weeklyweight gain of at least 1%, at least 2%, at least 3%, at least 4%, atleast 5%, at least 6%, at least 8%, at least 9%, at least 10%, at least11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3 to 5%greater than the average weekly weight gain of swine provided a dietthat does not include the synthetic oligosaccharide preparation.

E. Final Body Weight Gain

In some embodiments, providing an animal with a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition results in an increased final bodyweight gain than an animal provided feed without the syntheticoligosaccharide preparation. In some embodiments, providing an animalpopulation with a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition results inan increased average final body weight gain than an animal populationprovided feed without the synthetic oligosaccharide preparation.

In some embodiments, providing an animal or animal population with asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition results in a final body weightgain or average final body weight gain that is closer to the performancetarget maximum than an animal or animal population that is provided feedwithout the synthetic oligosaccharide preparation. The performancetarget maximum generally refers to the highest practical body weightgain observed for a given type of animal and breed under ideal growingconditions, ideal animal health, and ideal dietary nutrition.

In one embodiment, the final body weight gain is the quantity of weightan individual animal gains over a period of time. For example, in oneembodiment, the total body weight gain is the quantity of weight anindividual animal gains from 0 days of age until the final weight takenprior to processing of the animal, or the final weight taken on the dayof processing of the animal. For example, in one embodiment, the day 0to 28 total body weight gain for an animal is the quantity of weight anindividual animal gains from 0 days of age until 28 days of age.

In another embodiment, the average total body weight gain is thequantity of weight an individual animal gains over a period of time,averaged across an animal population. For example, in one embodiment,the average total body weight gain is the quantity of weight anindividual animal gains from 0 days of age until the final weight takenprior to processing of the animal, or the final weight taken on the dayof processing of the animal, averaged across the animal population. Inyet another embodiment, the average total body weight gain is thequantity of weight an animal population gains over a period of time,divided by the number of individual animals in the population. Forexample, in one embodiment, the average total body weight gain is thequantity of weight an animal population gains from 0 days of age untilthe final weight taken prior to processing of the animal population, orthe final weight taken on the day of processing of the animal, dividedby the number of individual animals in the population.

It should be understood that the values for total body weight gain andaverage total body weight gain can be further averaged. For example, theaverage total body weight gain for different populations of the sametype of animal may be averaged to obtain an average total body weightgain across populations.

In certain embodiments, the animal is poultry, and poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has a final body weight gain ofat least 3 kg, at least 2.5 kg, at least 2 kg, at least 1.5 kg, at least1 kg, between 1 to 3 kg, or between 1.5 to 2.5 kg. In one embodiment,poultry provided a synthetic oligosaccharide preparation, animal feedpre-mix, or animal feed composition has a final body weight gain of atleast 2 kg. In certain embodiments, poultry provided a syntheticoligosaccharide preparation, animal feed pre-mix, or animal feedcomposition has a final body weight gain of at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, between 1 to 10%, between2 to 8%, or between 3 to 5% greater than the final body weight gain ofpoultry provided a diet that does not include the syntheticoligosaccharide preparation. In certain embodiments, poultry provided asynthetic oligosaccharide preparation, animal feed pre-mix, or animalfeed composition has a final body weight gain of at least 0.01 kg, atleast 0.02 kg, at least 0.03 kg, at least 0.04 kg, at least 0.05 kg, atleast 0.06 kg, at least 0.07 kg, at least 0.08 kg, at least 0.09 kg, atleast 0.1 kg, between 0.01 to 0.1 kg, between 0.03 to 0.07 kg, orbetween 0.04 to 0.06 kg greater than the final body weight gain ofpoultry provided a diet that does not include the syntheticoligosaccharide preparation.

In certain embodiments, the animal is poultry, and poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has an average final bodyweight gain of at least 3 kg, at least 2.5 kg, at least 2 kg, at least1.5 kg, at least 1 kg, between 1 to 3 kg, or between 1.5 to 2.5 kg. Inone embodiment, poultry provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition has an average final body weight gain of at least 2 kg.In certain embodiments, poultry provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition has an average final body weight gain of at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than the averagefinal body weight gain of poultry provided a diet that does not includethe synthetic oligosaccharide preparation. In certain embodiments,poultry provided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition has anaverage final body weight gain of at least 0.01 kg, at least 0.02 kg, atleast 0.03 kg, at least 0.04 kg, at least 0.05 kg, at least 0.06 kg, atleast 0.07 kg, at least 0.08 kg, at least 0.09 kg, at least 0.1 kg,between 0.01 to 0.1 kg, between 0.03 to 0.07 kg, or between 0.04 to 0.06kg greater than the average final body weight gain of poultry provided adiet that does not include the synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the poultry is between 0to 14 days of age, between 15 to 28 days of age, between 29 to 35 daysof age, between 0 to 42 days of age, between 0 to 6 weeks of age, orbetween 0 to 6.5 weeks of age. In some embodiments, the starter phase is0 to 14 days of age, the grower phase is 15 to 28 days of age, and thefinisher phase is 29 to 35 days of age. In other embodiments, thestarter phase is 0 to 14 days of age, the grower phase is 15 to 35 daysof age, and the finisher phase is 36 to 42 days of age. In yet otherembodiments, the starter phase is 0 to 14 days of age, the grower phaseis 15 to 39 days of age, and the finisher phase is 40 to 46 days of age.It should be understood that the length of the starter phase, growingphase, and finisher phase for poultry may change depending on theintended use of the poultry, or the poultry product. For example, insome embodiments the length of the starter phase, grower phase, andfinisher phase may be different if the intended use of the poultry is asa broiler chicken, compared to processing for tray-pack chicken meat.

In some embodiments that may be combined with any of the foregoingembodiments, the poultry is an individual poultry, while in otherembodiments the poultry is a poultry population.

In certain embodiments, swine provided a synthetic oligosaccharidepreparation, swine nutritional composition, swine feed pre-mix, or swinefeed composition has a final body weight gain of at least 1%, at least2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 8%, atleast 9%, at least 10%, at least 11%, at least 12%, between 1 to 10%,between 2 to 8%, or between 3 to 5% greater than the final body weightgain of swine provided a diet that does not include the syntheticoligosaccharide preparation.

In certain embodiments, swine provided a synthetic oligosaccharidepreparation, swine nutritional composition, swine feed pre-mix, or swinefeed composition has an average final body weight gain of at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than the averagefinal body weight gain of swine provided a diet that does not includethe synthetic oligosaccharide preparation.

In some embodiments that may be combined with any of the foregoingembodiments, the swine is an individual swine, while in otherembodiments the swine is a swine population.

F. Yield of Animal Product

In certain embodiments, providing an animal with a syntheticoligosaccharide preparations, nutritional composition, animal feedpre-mix, or animal feed composition as described herein results in anincreased yield of animal product, as compared to an animal providedfeed that does not include the synthetic oligosaccharide preparation. Insome embodiments, the animal provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition yields at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, atleast 10%, between 1 to 10%, between 4 to 10%, between 6 to 10%, orbetween 2 to 8% more animal product compared to an animal provided feedthat does not include the synthetic oligosaccharide preparation. Forexample, in some embodiments, the animal product is the meat of theanimal, and an animal provided a synthetic oligosaccharide preparationas described herein yields a greater quantity of meat compared to ananimal that is not provided the oligosaccharide preparation. In someembodiments, providing an animal population the syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition results in an increased averageyield of animal product, as compared to an animal population providedfeed that does not include the synthetic oligosaccharide preparation. Insome embodiments, the average animal product yield is the quantity ofanimal product yielded from each individual animal, averaged across theanimal population.

In some embodiments, the animal product is the meat of an animal (e.g.,that may be sold to consumers, processed to produce a food product, orconsumed by a human). In certain embodiments, the animal is poultry, andthe animal product is a poultry eviscerated carcass, leg meat from apoultry eviscerated carcass, breast meat from a poultry evisceratedcarcass, drumstick meat from a poultry eviscerated carcass, fat from apoultry eviscerated carcass, breast meat from a poultry deboned carcass,or leg meat from a poultry deboned carcass. In other embodiments, theanimal is poultry, and the animal product is white meat, breast meatfilets, and breast meat tenders. In another embodiment, the animal ispoultry and the product is tray-pack chicken meat. In yet anotherembodiment, the animal is poultry and the product is whole bird withoutgiblets (WOG).

In some embodiments, the yield of animal product is the yield obtainedfrom an individual animal. In some embodiments, the average yield ofanimal product is the yield obtained from each individual animal in ananimal population, averaged across the population. In yet anotherembodiment, the average yield of animal product is the total yield ofanimal product yielded from an animal population, divided by the numberof individual animals in the animal population.

In some embodiments, the animal is poultry, the yield of leg meat from apoultry eviscerated carcass is at least 6%, at least 8%, at least 10%,at least 12%, between 6 to 12%, between 8 to 12%, between 10 to 18%,between 12 to 16%, or between 12 to 14% of live weight for poultryprovided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition. In certainembodiments, the yield of leg meat from a poultry eviscerated carcassfrom poultry provided a synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feed compositionas described herein is at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the average yield of legmeat from a poultry eviscerated carcass is at least 6%, at least 8%, atleast 10%, at least 12%, between 6 to 12%, between 8 to 12%, between 10to 18%, between 12 to 16%, or between 12 to 14% of live weight forpoultry provided a synthetic oligosaccharide preparations, nutritionalcomposition, animal feed pre-mix, or animal feed composition. In certainembodiments, the average yield of leg meat from a poultry evisceratedcarcass from poultry provided a synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feed compositionas described herein is at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the yield of breast meatfrom a poultry eviscerated carcass is at least 10%, at least 12%, atleast 15%, at least 16%, at least 18%, at least 20%, at least 22%, atleast 24%, at least 28%, between 10 to 18%, between 12 to 16%, between18 to 29%, between 20 to 27%, or between 20 to 25% of live weight forpoultry provided a synthetic oligosaccharide preparations, nutritionalcomposition, animal feed pre-mix, or animal feed composition. In certainembodiments, the yield of breast meat from a poultry eviscerated carcassfrom poultry provided a synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feed compositionas described herein is at least 1%, at least 2%, at least 3%, at least4%, at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the average yield ofbreast meat from a poultry eviscerated carcass is at least 10%, at least12%, at least 15%, at least 16%, at least 18%, at least 20%, at least22%, at least 24%, at least 28%, between 10 to 18%, between 12 to 16%,between 18 to 29%, between 20 to 27%, or between 20 to 25% of liveweight for poultry provided a synthetic oligosaccharide preparations,nutritional composition, animal feed pre-mix, or animal feedcomposition. In certain embodiments, the average yield of breast meatfrom a poultry eviscerated carcass from poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition as described herein is at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than for poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In some embodiments, the animal is poultry, and the yield of drumstickmeat from a poultry eviscerated carcass is at least 5%, at least 7%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between5 to 14%, between 7 to 10%, between 7 to 15%, between 9 to 13%, orbetween 9 to 11% of live weight for poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, the yieldof drumstick meat from a poultry eviscerated carcass from poultryprovided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition asdescribed herein is at least 1%, at least 2%, at least 3%, at least 4%,at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the average yield ofdrumstick meat from a poultry eviscerated carcass is at least 5%, atleast 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least12%, between 5 to 14%, between 7 to 10%, between 7 to 15%, between 9 to13%, or between 9 to 11% of live weight for poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, the averageyield of drumstick meat from a poultry eviscerated carcass from poultryprovided a synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition asdescribed herein is at least 1%, at least 2%, at least 3%, at least 4%,at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the yield of breast meatfrom a poultry deboned carcass is at least 14%, at least 16%, at least18%, at least 20%, at least 22%, at least 24%, between 14 to 16%,between 18 to 30%, between 20 to 28%, or between 20 to 26% of liveweight for poultry provided a synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feedcomposition. In certain embodiments, the yield of breast meat from apoultry deboned carcass from poultry provided an oligosaccharidepreparation, animal feed pre-mix, or animal feed composition asdescribed herein is at least 1%, at least 2%, at least 3%, at least 4%,at least 5%, at least 6%, at least 8%, at least 9%, at least 10%, atleast 11%, at least 12%, between 1 to 10%, between 2 to 8%, or between 3to 5% greater than for poultry provided a diet that does not include thesynthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the average yield ofbreast meat from a poultry deboned carcass is at least 14%, at least16%, at least 18%, at least 20%, at least 22%, at least 24%, between 14to 16%, between 18 to 30%, between 20 to 28%, or between 20 to 26% oflive weight for poultry provided a synthetic oligosaccharidepreparations, nutritional composition, animal feed pre-mix, or animalfeed composition. In certain embodiments, the average yield of breastmeat from a poultry deboned carcass from poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition as described herein is at least 1%,at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than for poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In some embodiments, the animal is poultry, and the yield of leg meatfrom a poultry deboned carcass is at least 6%, at least 8%, at least10%, at least 12%, at least 14%, at least 16%, at least 18%, between 6to 18%, between 8 to 16%, between 12 to 21%, between 14 to 19%, orbetween 14 to 17% of live weight for poultry provided a syntheticoligosaccharide preparations, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, the yieldof leg meat from a poultry deboned carcass from poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition as described herein is at least1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than for poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In some embodiments, the animal is poultry, and the average yield of legmeat from a poultry deboned carcass is at least 6%, at least 8%, atleast 10%, at least 12%, at least 14%, at least 16%, at least 18%,between 6 to 18%, between 8 to 16%, between 12 to 21%, between 14 to19%, or between 14 to 17% of live weight for poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition. In certain embodiments, theaverage yield of leg meat from a poultry deboned carcass from poultryprovided an oligosaccharide preparation, animal feed pre-mix, or animalfeed composition as described herein is at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, between 1 to 10%, between2 to 8%, or between 3 to 5% greater than for poultry provided a dietthat does not include the synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the yield of fat from apoultry eviscerated carcass is at least 0.1%, at least 0.2%, at least0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, atleast 0.8%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%, atleast 1.6%, between 0.1 to 2%, between 0.2 to 1%, between 0.5 to 2%, orbetween 0.3 to 0.7% of live weight for poultry provided a syntheticoligosaccharide preparation, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, the yieldof fat from a poultry eviscerated carcass from poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition as described herein is at least1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than for poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In some embodiments, the animal is poultry, and the average yield of fatfrom a poultry eviscerated carcass is at least 0.1%, at least 0.2%, atleast 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%,at least 0.8%, at least 0.9%, at least 1%, at least 1.2%, at least 1.4%,at least 1.6%, between 0.1 to 2%, between 0.2 to 1%, between 0.5 to 2%,or between 0.3 to 0.7% of live weight for poultry provided a syntheticoligosaccharide preparations, nutritional composition, animal feedpre-mix, or animal feed composition. In certain embodiments, the averageyield of fat from a poultry eviscerated carcass from poultry provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition as described herein is at least1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, atleast 8%, at least 9%, at least 10%, at least 11%, at least 12%, between1 to 10%, between 2 to 8%, or between 3 to 5% greater than for poultryprovided a diet that does not include the synthetic oligosaccharidepreparation.

In some embodiments, the animal is poultry, and the yield of a poultryeviscerated carcass is at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, between 50 to 95%, between 60 to 85%, or between 65 to 75% oflive weight for poultry provided a synthetic oligosaccharidepreparations, nutritional composition, animal feed pre-mix, or animalfeed composition. In certain embodiments, the yield of a poultryeviscerated carcass from poultry provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition as described herein is at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, between 1 to 10%, between2 to 8%, or between 3 to 5% greater than for poultry provided a dietthat does not include the synthetic oligosaccharide preparation.

In some embodiments, the animal is poultry, and the average yield of apoultry eviscerated carcass is at least 50%, at least 55%, at least 60%,at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, between 50 to 95%, between 60 to 85%, or between 65 to 75% oflive weight for poultry provided a synthetic oligosaccharidepreparations, nutritional composition, animal feed pre-mix, or animalfeed composition. In certain embodiments, the average yield of a poultryeviscerated carcass from poultry provided a synthetic oligosaccharidepreparation, nutritional composition, animal feed pre-mix, or animalfeed composition as described herein is at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 6%, at least 8%, at least9%, at least 10%, at least 11%, at least 12%, between 1 to 10%, between2 to 8%, or between 3 to 5% greater than for poultry provided a dietthat does not include the synthetic oligosaccharide preparation.

Methods for deboning a poultry carcass are well known to one skilled inthe art of poultry processing. It should be understood that meat yieldedfrom poultry may be measured, for example, as the ratio of the mass ofrecovered meat to the final weight of the bird prior to processing. Insome embodiments, the animal is poultry, and the poultry is at least 35days old, at least 42 days old, at least 6 weeks old, at least 6.5 weeksold before the poultry is processed to produce a poultry evisceratedcarcass, poultry deboned carcass, white meat, breast meat filets, andbreast meat tenders, tray-pack chicken meat, whole bird without giblets(WOG), or meat as described above.

In other embodiments, the animal is poultry, and the animal product iseggs. In some embodiments, the animal is swine, and the swine product isthe meat of swine (e.g., that may be sold to consumers, processed toproduce a food product, or consumed by a human). In some embodiments,the yield of swine product is the yield obtained from an individualswine. In some embodiments, the average yield of swine product is theyield obtained from each individual swine in a swine population,averaged across the population. In yet another embodiment, the averageyield of swine product is the total yield of swine product yielded fromswine population, divided by the number of individual swine in the swinepopulation.

In certain embodiments, an animal or animal population provided asynthetic oligosaccharide preparation, nutritional composition, animalfeed pre-mix, or animal feed composition has a higher average dailyweight gain, higher average weekly weight gain, higher final body weightgain, higher average final body weight gain, or increased average yieldof animal product, or any combinations thereof, than an animal or animalpopulation provided a diet that does not include the syntheticoligosaccharide preparation, but which does include one or moreantibiotics, one or more ionophores, soluble corn fiber, modified wheatstarch, or yeast mannan, or any combinations thereof.

A person of skill in the art would recognize that the maximumtheoretical weight gain may be different for different types of animalsand may be different for different breeds of the same type of animal(for example, different types of broiler chickens, or different types ofswine).

A person of skill in the art would recognize that the maximumtheoretical weight gain may be different for different types of animalsand may be different for different breeds of the same type of animal(for example, different types of broiler chickens, or different types ofswine).

In some embodiments, the animal is poultry. In some embodiments that maybe combined with any of the foregoing embodiments, the poultry is anindividual poultry, while in other embodiments the poultry is a poultrypopulation. In other embodiments, the animal is swine. In someembodiments that may be combined with any of the foregoing embodiments,the swine is an individual swine, while in other embodiments the swineis a swine population.

G. Feed Intake

In certain embodiments, providing an animal with a syntheticoligosaccharide preparation nutritional composition, animal feedpre-mix, or animal feed composition as described herein results in anincreased average daily feed intake, as compared to an animal providedfeed that does not include the synthetic oligosaccharide preparation.

Average daily feed intake (ADFI) refers to the average mass of feedconsumed by an animal over a specified period of time. In certainembodiments, the average daily feed intake is measured by dispensing aknown mass of feed to a group of a fixed number of animals, allowing theanimals in the group to consume the dispensed feed freely (ad libidum)for a specified number of days, weighing the mass of unconsumed feed atthe end of the period, and calculating the average daily feed intake(ADFI) as the difference between the dispensed feed mass minus theresidual feed mass, divided by the number of animals in the group, anddivided by the number of days in the period. In other embodiments, theaverage daily feed intake may be corrected for any animals that die orare culled from the group, using methods that are known to one skilledin the art.

In some embodiments, the animal is poultry, and the animal feedcomposition is poultry feed, wherein the synthetic oligosaccharidepreparation, poultry feed pre-mix, or poultry feed composition feedincreases average daily feed intake by up to about 10%, or about 5%, orbetween 1% and 10%, between 2% and 10%, between 3% and 10%, between 4%and 10%, between 5% and 10%, between 2% and 5%, between 2% and 6%,between 2% and 7%, between 2% and 8%, between 2% and 9%, or between 1%and 5%, when fed to poultry as compared to poultry fed a feedcomposition without the synthetic oligosaccharide preparation.

In certain embodiments, the poultry suffers from a disease or is raisedin a challenged environment, wherein the synthetic oligosaccharidepreparation, poultry nutritional composition, poultry feed pre-mix, orpoultry feed composition increases average daily feed intake by up toabout 30%, about 25%, about 20%, about 15%, about 10%, or about 5%, orbetween 1% and 30%, between 5% and 30%, between 10% and 30%, between 5%and 20%, between 10% and 20%, between 1% and 20%, between 1% and 15%,between 1% and 10%, between 2% and 10%, between 3% and 10%, between 4%and 10%, between 5% and 10%, between 2% and 5%, between 2% and 6%,between 2% and 7%, between 2% and 8%, between 2% and 9%, or between 1%and 5%, when fed to poultry as compared to poultry fed a feedcomposition without the synthetic oligosaccharide preparation.

In some embodiments that may be combined with the foregoing, the animalis swine, and the animal feed composition is swine feed, wherein theoligosaccharide preparation, swine nutritional composition, swine feedpre-mix, or swine feed composition increases average daily feed intakeby up to about 15%, about 10%, or about 5%, or between 1% and 15%,between 2% and 15%, between 3% and 15%, between 4% and 15%, between 5%and 15%, between 10% and 15%, between 1% and 10%, between 2% and 10%,between 3% and 10%, between 4% and 10%, between 5% and 10%, between 2%and 5%, between 2% and 6%, between 2% and 7%, between 2% and 8%, between2% and 9%, or between 1% and 5%, when fed to swine as compared to swinefed a feed composition without the synthetic oligosaccharidepreparation.

In certain embodiments, the swine suffers from a disease or is raised ina challenged environment, wherein the synthetic oligosaccharidepreparation, swine nutritional composition, swine feed pre-mix, or swinefeed composition increases average daily feed intake by up to about 40%,about 35% about 30%, about 25%, about 20%, about 15%, about 10%, orabout 5%, or between 1% and 40%, between 5% and 40%, between 10% and40%, between 15% and 40%, between 20% and 40%, between 25% and 40%,between 30% and 40%, between 1% and 30%, between 5% and 30%, between 10%and 30%, between 5% and 20%, between 10% and 20%, between 1% and 20%,between 1% and 15%, between 1% and 10%, between 2% and 10%, between 3%and 10%, between 4% and 10%, between 5% and 10%, between 2% and 5%,between 2% and 6%, between 2% and 7%, between 2% and 8%, between 2% and9%, or between 1% and 5%, when fed to swine as compared to swine fed afeed composition without the synthetic oligosaccharide preparation.

The methods of enhancing growth of an animal or animal populationdescribed herein include providing an oligosaccharide preparation,animal feed pre-mix, or animal feed to the animal or animal population.The oligosaccharide preparation, animal feed pre-mix, or animal feed maybe provided in any suitable form, to any suitable type of animal, usingany suitable feeding schedule to enhance the growth of the animal oranimal population.

VIII. Animals

The synthetic oligosaccharide preparation, nutritional composition,animal feed pre-mix, or the animal feed composition may be provided toany suitable animal. In some embodiments, the animal is monogastric. Itis generally understood that a monogastric animal has a single-chamberedstomach. In other embodiments, the animal is a ruminant. It is generallyunderstood that a ruminant has a multi-chambered stomach. In someembodiments, the animal is a ruminant in the pre-ruminant phase.Examples of such ruminants in the pre-ruminant phase include nurserycalves.

In some embodiments, the animal is livestock. In some embodiments, theanimal is a companion animal. In some embodiments, the animal ispoultry. Examples of poultry include chicken, duck, turkey, goose,quail, or Cornish game hen. In one variation, the animal is a chicken.In some embodiments, the poultry is a layer hen, a broiler chicken, or aturkey. In other embodiments, the animal is a mammal, including, forexample, a cow, a pig, a goat, a sheep, a deer, a bison, a rabbit, analpaca, a llama, a mule, a horse, a reindeer, a water buffalo, a yak, aguinea pig, a rat, a mouse, an alpaca, a dog, a cat, or a human. In onevariation, the animal is a cow. In another variation, the animal is apig.

The animal feed composition may also be used in aquaculture. In someembodiments, the animal is an aquatic animal. Examples of aquaticanimals may include a trout, a salmon, a bass, a tilapia, a shrimp, anoyster, a mussel, a clam, a lobster, or a crayfish. In one variation,the animal is a fish.

In some embodiments, the animal is a fish (e.g. salmon, tilapia,tropical fish), poultry (e.g. chicken, turkey), seafood (e.g. shrimp),sheep, cow, cattle, buffalo, bison, pig (e.g. nursery pig,grower/finisher pig), cat, dog, rabbit, goat, guinea pig, donkey, camel,horse, pigeon, ferret, gerbil, hamster, mouse, rat, bird, or human.

IX. Administration

In some embodiments, administration comprises providing a syntheticoligosaccharide preparation, a nutritional composition, or an animalfeed composition described herein, to an animal such that the animal mayingest the synthetic oligosaccharide preparation, the nutritionalcomposition, or the animal feed composition at will. In suchembodiments, the animal ingests some portion of the syntheticoligosaccharide preparation, the nutritional composition, or the animalfeed composition.

The synthetic oligosaccharide preparation, nutritional composition,animal feed pre-mix, or animal feed composition may be provided to theanimal on any appropriate schedule. In some embodiments, the animal isprovided the synthetic oligosaccharide preparation, nutritionalcomposition, animal feed pre-mix, or animal feed composition on a dailybasis, on a weekly basis, on a monthly basis, on an every other daybasis, for at least three days out of every week, or for at least sevendays out of every month. In some embodiments, the animal is provided theoligosaccharide preparation, animal feed pre-mix, or animal feedcomposition during certain diet phases.

For example, some animals are provided a starter diet between 0 to 14days of age. In other embodiments, an animal is provided a grower dietbetween 15 to 28 days of age, between 15 to 35 days of age, or between15 to 39 days of age. In still other embodiments, an animal is provideda finisher diet between 29 to 35 days of age, between 36 to 42 days ofage, or between 40 to 46 days of age.

In certain embodiments, the synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feed compositionis provided to the animal during the starter diet phase, the grower dietphase, or the finisher diet phase, or any combinations thereof.

In certain embodiments, the animal is poultry, and the poultry isprovided a starter diet between 0 to 15 days of age, a grower dietbetween 16 to 28 days of age, and a finisher diet between 29 to 35 daysof age. In other embodiments, the animal is poultry, and the poultry isprovided a starter diet between 0 to 14 days of age, a grower dietbetween 15 to 35 days of age, and a finisher diet between 36 to 42 daysof age. In still other embodiments, the animal is poultry, and thepoultry is provided a starter diet between 0 to 14 days of age, a growerdiet between 15 to 39 days of age, and a finisher diet between 20 to 46days of age.

In some embodiments, the synthetic oligosaccharide preparation,nutritional composition, animal feed pre-mix, or animal feed compositionis provided to the poultry during the starter diet phase, the growerdiet phase, or the finisher diet phase, or any combinations thereof

The oligosaccharide preparations described herein may be fed toindividual animals or an animal population. For example, in onevariation where the animal is poultry, the oligosaccharide preparationsmay be fed to an individual poultry or a poultry population.

The synthetic oligosaccharide preparation, nutritional composition,animal feed pre-mix, or the animal feed composition may be provided toan animal in any appropriate form, including, for example, in solidform, in liquid form, or a combination thereof. In certain embodiments,the oligosaccharide preparation or the animal feed composition is aliquid, such as a syrup or a solution. In other embodiments, theoligosaccharide preparation, animal feed pre-mix, or the animal feedcomposition is a solid, such as pellets or powder. In yet otherembodiments, the oligosaccharide preparation, animal feed pre-mix, orthe animal feed composition may be fed to the animal in both liquid andsolid components, such as in a mash.

EXAMPLES Example 1 Synthesis of a Gluco-Galacto-OligosaccharidePreparation

Synthesis of a gluco-galacto-oligosaccharide preparation was performedin a three-liter reaction vessel using catalyst loadings, reactiontimes, and reaction temperatures that were selected to enable suitableproduction at the kg scale.

D-glucose monohydrate (825.16 g), D-lactose monohydrate (263.48 g) and2-pyridinesulfonic acid (1.0079 g, Sigma-Aldrich, St. Louis, US) wereadded to a three-liter, three-neck round bottom flask with a center29/42 ground glass joint and two 24/40 side ground glass joints. A 133mm Teflon stirring blade was affixed to a glass stir shaft using PTFEtape. The stir rod was secured through the center point using a Teflonbearing adapter and attached to an overhead high-torque mechanical mixervia flexible coupler. The flask was secured inside a hemisphericalelectric heating mantle operated by a temperature control unit via aJ-type wand thermocouple inserted through a rubber septum in one of theside ports. The tip of the thermocouple was adjusted to reside withinthe reaction mixture with several mm clearance above the mixing element.A secondary temperature probe connected to an auxiliary temperaturemonitor was also inserted and secured by the same means. The second sideport of the flask was equipped with a reflux condenser cooled by awater-glycol mixture maintained below 4° C. by a recirculating bathchiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. When the reaction mixture reached120° C., the reflux condenser was re-positioned into a distillationconfiguration, with the distillated collected in a 250 mL round bottomflask placed in an ice bath. The mixture was maintained at 130° C. withcontinuous mixing for 6 hours, after which the thermocouple box waspowered off. The distillation apparatus was removed and 390 g of 60° C.distilled water was gradually added into the three-neck flask. Theresulting mixture was left to stir at 40 RPM for 10 hours. Approximately1,250 g of a viscous, light-amber material was collected and measured byrefractive index to have a concentration of 71.6 Brix.

The final water content of the reactor product was measured by KarlFisher titration for a representative aliquot of the reactor contentsdrawn at the end of the reaction. At a reaction temperature of 130° C.,the water content of the reaction product was determined to be 5.8 wt %water on an as-is basis.

Example 2 Synthesis of a Gluco-Oligosaccharide Preparation

Synthesis of a gluco-oligosaccharide preparation was performed in athree-liter reaction vessel using catalyst loadings, reaction times, andreaction temperatures that were selected to enable suitable productionat the kg scale.

D-glucose monohydrate (1,150 g) was added to a three-liter, three-neckround bottom flask with one center 29/42 ground glass joint and two side24/40 ground glass joints. A 133 mm Teflon stirring blade was affixed toglass stir shaft using PTFE tape. The stir rod was secured through thecenter port of the flask using a Teflon bearing adapter and attached toan overhead high-torque mechanical mixer via flex coupling. The flaskwas secured inside a hemispherical electric heating mantle operated by atemperature control unit via a J-type wand thermocouple inserted througha rubber septum in one of the side ports. The tip of the thermocouplewas adjusted to reside within the reaction mixture with several mmclearance above the mixing element. A secondary temperature probeconnected to an auxiliary temperature monitor was also inserted andsecured by the same means. The second side port of the flask wasequipped with a reflux condenser cooled by a water-glycol mixturemaintained below 4 C by a recirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. When the reaction temperatureincreased to between 120° C. and 130° C., (+)-Camphor-10-sulfonic acid(1.16 g, Sigma-Aldrich, St. Louis) was added to the three-neck flask andthe apparatus was switched from a reflux condenser to a distillationconfiguration with a round bottom collection flask placed in an icebath. This setup was maintained for 1 and a half hours, after which thethermocouple box was powered off, the distillation apparatus wasremoved, and 390 g of 23 degrees C. distilled water was gradually addedinto the three-neck flask. The resulting mixture was left to stir at 40rpm for 10 hours until the moment of collection. Approximately 1300 g ofa viscous, dark-amber material was collected and measured to have aconcentration of 72.6 brix.

Example 3 Synthesis of a Gluco-Galacto-Manno-Oligosaccharide Preparation

Synthesis of a gluco-galacto-manno-oligosaccharide preparation wasperformed in a three-liter reaction vessel using catalyst loadings,reaction times, and reaction temperatures that were selected to enablesuitable production at the kg scale.

The gluco-galacto-manno-oligosaccharide was prepared as two separatecomponents synthesized in separate reaction vessels that wereindependently collected. Each synthesis used different startingreactants but followed the same procedure and methods to completion. Thefinal gluco-galacto-manno-oligosaccharide was a homogeneous syrup formedfrom the mixing of both synthesis products.

For the synthesis of the first component, 990.54 g of glucosemonohydrate, 105.58 g of lactose monohydrate and 1.00 g of2-pyridinesulfonic acid were added to a three-liter, three-neck roundbottom flask with one center 29/42 ground joint flanked by two 24/40ground joints. A 133 mm Teflon stirring blade was affixed to a 440 mmglass stir shaft using PTFE tape. The stir rod was secured through thecenter point using a Teflon bearing adapter and attached to an overheadhigh-torque mechanical mixer via flexible coupler. The flask was placedinside a hemispherical electric heating mantle operated by a temperaturecontrol unit via a J-type wand thermocouple inserted through a rubberseptum in one of the side ports. The tip of the thermocouple wasadjusted to reside within the reaction mixture with several mm clearanceabove the mixing element. A secondary temperature probe connected to anauxiliary temperature monitor was also inserted and secured by the samemeans. The second side port of the flask was equipped with a refluxcondenser cooled by a water-glycol mixture maintained below 4° C. by arecirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. Once a temperature control boxreading between 120 C and 130° C. was observed, the apparatus wasswitched from a reflux condenser to a distillation configuration with around bottom collection flask placed in an ice bath. This setup wasmaintained for approximately 6 hours and 10 minutes, after which theheating mantle was powered off, the distillation apparatus was removed,and 390 g of 60° C. distilled water was gradually added into thethree-neck flask. The resulting mixture was left to stir at 40 rpm for10 hours until the moment of collection. Approximately 1250 g of aviscous, light-amber material was collected and measured by refractiveindex to have a concentration of 73.1 Brix.

For the synthesis of the second component, 825.04 g of glucosemonohydrate, 251.16 g of pure mannose from wood, 25.10 g distilledwater, and 1.00 g of 2-pyridinesulfonic acid were added to athree-liter, three-neck round bottom flask with one center 29/42 groundjoint flanked by two 24/40 ground joints. The remainder of the secondcomponent's synthesis followed the same procedure and methods as thoseof the first, until the moment of collection. Approximately 1250 g of aviscous, dark-amber material was collected and measured to have aconcentration of 72.3 brix.

The entirety of the first and second components were transferred into asuitably sized HDPE container and mixed thoroughly by hand untilhomogenous. The final syrup mixture was approximately 2.5 kg, dark-amberin color, viscous and was measured to have a concentration ofapproximately 72 brix.

Example 4 Synthesis of a Gluco-Manno-Oligosaccharide Preparation

Synthesis of a gluco-oligosaccharide preparation was performed in athree-liter reaction vessel using catalyst loadings, reaction times, andreaction temperatures that were selected to enable suitable productionat the kg scale.

A gluco-manno-oligosaccharide was prepared as two separate componentssynthesized in separate reaction vessels that were independentlycollected. Each synthesis used different starting reactants but followedthe same procedure and methods to completion. The finalgluco-manno-oligosaccharide was a homogeneous syrup formed from themixing of both synthesis products.

For the synthesis of the first component, 1264.80 g of glucosemonohydrate was added to a three-liter, three-neck round bottom flaskwith one center 29/42 ground joint flanked by two 24/40 ground joints. A133 mm Teflon stirring blade was affixed to a 440 mm glass stir shaftusing PTFE tape. The stir rod was secured through the center point usinga Teflon bearing adapter and attached to an overhead high-torquemechanical mixer via flexible coupler. The flask was placed inside ahemispherical electric heating mantle operated by a temperature controlunit via a J-type wand thermocouple inserted through a rubber septum inone of the side ports. The tip of the thermocouple was adjusted toreside within the reaction mixture with several mm clearance above themixing element. A secondary temperature probe connected to an auxiliarytemperature monitor was also inserted and secured by the same means. Thesecond side port of the flask was equipped with a reflux condensercooled by a water-glycol mixture maintained below 4° C. by arecirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. Once a temperature control boxreading between 120° C. and 130° C. was observed, 1.15 g of(+)-camphor-10-sulfonic acid was added to the three-neck flask and theapparatus was switched from a reflux condenser to a distillationconfiguration with a round bottom collection flask placed in an icebath. This setup was maintained for approximately 1 hour, after whichthe thermocouple box was powered off, the distillation apparatus wasremoved, and 390 g of 23° C. distilled water was gradually added intothe three-neck flask. The resulting mixture was left to stir at 40 rpmfor 10 hours until the moment of collection. Approximately 1350 g of aviscous, light-amber material was collected and measured to have aconcentration of 71.8 brix.

For the synthesis of the second component, 949.00 g of glucosemonohydrate, 288.00 g of pure mannose from wood, 27.94 g distilledwater, and 1.15 g of 2-pyridinesulfonic acid were added to athree-liter, three-neck round bottom flask with one center 29/42 groundjoint flanked by two 24/40 ground joints. The remainder of the secondcomponent's synthesis followed the same procedure and methods as thoseof the first until the moment of collection, except(+)-camphor-10-sulfonic acid was not added as the reflux condenser wasswitched to a distillation configuration and the resulting setup wasmaintained for approximately 6 hours. Approximately 1350 g of a viscous,dark-amber material was collected and measured to have a concentrationof 72.0 brix.

The entirety of the first and second components were transferred into asuitably sized HDPE container and mixed thoroughly by hand untilhomogenous. The final syrup mixture was approximately 2.7 kg, dark-amberin color, viscous and was measured by refractive index to have aconcentration of approximately 72 Brix.

Example 5 Synthesis of a Gluco-Manno-Oligosaccharide Preparation

Kilogram scale production of the oligosaccharide preparation wasperformed in a three-liter reaction vessel using catalyst loadings,reaction times, and reaction temperatures found to be suitable forproduction at the 1 kg scale.

A gluco-manno-oligosaccharide was prepared as two separate componentssynthesized in separate reaction vessels that were independentlycollected. Each synthesis used different starting reactants but followedthe same procedure and methods to completion. The finalgluco-manno-oligosaccharide was a homogeneous syrup formed from themixing of both synthesis products.

For the synthesis of the first component, 1261.00 g of glucosemonohydrate and 1.15 g of 2-pyridinesulfonic acid were added to athree-liter, three-neck round bottom flask with one center 29/42 groundjoint flanked by two 24/40 ground joints. A 133 mm Teflon stirring bladewas affixed to a 440 mm glass stir shaft using PTFE tape. The stir rodwas secured through the center point using a Teflon bearing adapter andattached to an overhead high-torque mechanical mixer via flexiblecoupler. The flask was secured inside a hemispherical electric heatingmantle operated by a temperature control unit via a J-type wandthermocouple inserted through a rubber septum in one of the side ports.The tip of the thermocouple was adjusted to reside within the reactionmixture with several mm clearance above the mixing element. A secondarytemperature probe connected to an auxiliary temperature monitor was alsoinserted and secured by the same means. The second side port of theflask was equipped with a reflux condenser cooled by a water-glycolmixture maintained below 4° C. by a recirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. Once a temperature control boxreading between 120° C. and 130° C. was observed, the apparatus wasswitched from a reflux condenser to a distillation configuration with around bottom collection flask placed in an ice bath. This setup wasmaintained for approximately 6 hours, after which the thermocouple boxwas powered off, the distillation apparatus was removed, and 390 g of23° C. distilled water was gradually added into the three-neck flask.The resulting mixture was left to stir at 40 rpm for 10 hours until themoment of collection. Approximately 1250 g of a viscous, light-ambermaterial was collected and measured to have a concentration of 73.5brix.

For the synthesis of the second component, 949.00 g of glucosemonohydrate, 288.00 g of pure mannose from wood, 28.94 g distilledwater, and 1.15 g of 2-pyridinesulfonic acid were added to athree-liter, three-neck round bottom flask with one center 29/42 groundjoint flanked by two 24/40 ground joints. The remainder of the secondcomponent's synthesis followed the same procedure and methods as thoseof the first until the moment of collection. Approximately 1250 g of aviscous, dark-amber material was collected and measured to have aconcentration of 73.3 brix.

The entirety of the first and second components were transferred into asuitably sized HDPE container and mixed thoroughly by hand untilhomogenous. The final syrup mixture was approximately 2.5 kg, dark-amberin color, viscous and was measured to have a concentration ofapproximately 73 brix.

Example 6 Synthesis of a Gluco-Galacto-Oligosaccharide Preparation

Kilogram scale production of the oligosaccharide preparation wasperformed in a three-liter reaction vessel using catalyst loadings,reaction times, and reaction temperatures found to be suitable forproduction at the 1 kg scale.

A 3 L three-neck flask was equipped with an overhead mixer connected viaa 10 mm diameter glass stir-shaft to a 14 cm crescent-shaped mixingelement. The mixing element was positioned with approximately 5 mmclearance from the walls of the flask. The flask was heated via ahemispherical electric heating mantle powered by a temperature controlunit connected to a wand-type thermocouple probe inserted into thereaction flask. The thermocouple probe was placed to provide 5-10 mmclearance above the mixing element. The flask was charged with 576 gramsof food-grade dextrose monohydrate and 577 grams of food-gradeD-galactose monohydrate and heated to approximately 115° C. to obtain amolten sugar syrup. Once the syrup was obtained, the flask was fittedwith a jacketed reflux condenser cooled to 4° C. by circulating chilledglycol/water and the temperature. 31 grams of Dowex Marathon C (moisturecontent 0.48 g H₂O/g resin) were added to the mixture to form a stirredsuspension. The condenser was repositioned into distillationconfiguration and the suspension was heated to 145° C.

A mixing rate of approximately 80 RPM and a temperature of 145° C. wasmaintained for 3.8 hours, after which the set point on the temperaturecontrol unit was reduced to 80° C. and 119 mL of 60° C. deionized waterwas gradually added to the flask to obtain a dark amber syrup containingresidual Dowex resin. The resulting suspension was further diluted to 60Brix, cooled to room temperature and vacuum filtered through a 0.45micron filter to remove the resin. 1,200 grams of light-amber syrup at60 Brix concentration was obtained.

Example 7 Synthesis of a Gluco-Oligosaccharide Preparation

Kilogram scale production of the oligosaccharide preparation wasperformed in a three-liter reaction vessel using catalyst loadings,reaction times, and reaction temperatures found to be suitable forproduction at the 1 kg scale.

A 3 L three-neck flask was equipped with an overhead mixer connected viaa 10 mm diameter glass stir-shaft to a 14 cm crescent-shaped mixingelement. The mixing element was positioned with approximately 5 mmclearance from the walls of the flask. The flask was heated via ahemispherical electric heating mantle powered by a temperature controlunit connected to a wand-type thermocouple probe inserted into thereaction flask. The thermocouple probe was placed to provide 5-10 mmclearance above the mixing element. The flask was gradually charged with1,148 grams of food-grade dextrose monohydrate and heated toapproximately 115° C. to obtain a molten sugar syrup. Once the syrup wasobtained, the flask was fitted with a jacketed distillation condensercooled to 4° C. by circulating chilled glycol/water. The reactiontemperature was gradually increased to 145° C. Once the temperature wasobtained and stable, 31 grams of Dowex Marathon C (moisture content 0.48g H₂O/g resin) was added to the mixture and a mixing rate ofapproximately 80 RPM and a temperature of 145° C. was maintained for 3.8hours.

After 3.8 hours, the set point on the temperature control unit wasreduced to 80° C. and 119 mL of 60° C. deionized water was graduallyadded to the flask to obtain a dark amber syrup containing residualDowex resin. The resulting suspension was further diluted to 60 Brix,cooled to room temperature and vacuum filtered through a 0.45 micronfilter to remove the resin. 1,113 grams of dark-ambergluco-oligosaccharide syrup at 60 Brix concentration was obtained.

Example 8 Single-Pot Syntheses of Oligosaccharide Preparations

A single pot (single component) synthesis of the oligosaccharide fromExample 3 was demonstrated at 300 gram scale in a one-liter reactionvessel using catalyst loadings, reaction times, and reactiontemperatures found to be suitable for the single pot reaction.

30 g of food-grade D-glucose monohydrate from corn, 37.50 g of foodgrade D-mannose from wood, 15.60 g of food-grade D-lactose monohydrate,3.96 g of distilled water and 0.270 g of 2-pyridinesulfonic acid(Sigma-Aldrich, St. Louis) were added to a one-liter, three-neck roundbottom flask with one center 29/42 ground joint flanked by two 24/40ground joints. A Teflon stirring blade was affixed to a 220 mm glassstir shaft using PTFE tape. The stir rod was secured through the centerpoint using a Teflon bearing adapter and attached to an overheadhigh-torque mechanical mixer via flexible coupler. The flask was securedinside a hemispherical electric heating mantle operated by a temperaturecontrol unit via a J-type wand thermocouple inserted through a rubberseptum in one of the side ports. The tip of the thermocouple wasadjusted to reside within the reaction mixture with several mm clearanceabove the mixing element. A secondary temperature probe connected to anauxiliary temperature monitor was also inserted and secured by the samemeans. The second side port of the flask was equipped with a refluxcondenser cooled by a water-glycol mixture maintained below 4° C. by arecirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. Once a temperature control boxreading between 120° C. and 130° C. was observed, the apparatus wasswitched from a reflux condenser to a distillation configuration with around bottom collection flask placed in an ice bath. The mixture wasmaintained at 130° C. with continuous stirring for approximately 5 hoursand 40 minutes, after which the heating mantle and distillationapparatus was removed. Approximately 40 g of 23° C. distilled water wasgradually added into the three-neck flask. The resulting mixture wasleft to stir at 40 rpm for 10 hours until the moment of collection.Approximately 389 g of a viscous, dark-amber material was collected andmeasured to have a concentration of 67.0 brix. Consistency with theoligosaccharide preparation from Example 3 was confirmed by SECchromatography and 2D HS QC NMR spectroscopy.

Example 9 Characterization of Oligosaccharide Preparations

The methods and procedures from Examples 1-8 were used to preparereplicate batches and blends of the oligosaccharides of Examples 1-7. Inpreparing the various batches of Examples 9.1-9.7, both multi-pot(multi-component) and single-pot variants of the respective syntheticschemes were employed. The resulting materials were analyzed by HPLCSize Exclusion Chromatography (SEC) to characterize the molecular weightdistribution, LC-MS/MS analysis to quantify the DP2 anhydro-sugarcontent, and 2D ¹H, ¹³C-HSQC NMR to fingerprint the molecular structureof the corresponding oligosaccharide preparations. Materials wereprepared as follows:

Example 9.1: eleven batches of the oligosaccharide preparation fromExample 1 were prepared and blended into four separate lots to produceoligosaccharide 9.1.

Example 9.2: seven batches of the oligosaccharide preparation fromExample 2 were prepared and blended into two separate lots to produceoligosaccharide 9.2.

Example 9.3: twelve batches of the oligosaccharide preparation fromExample 3 were prepared and blended into five separate lots to produceoligosaccharide 9.3.

Example 9.4: four batches of the oligosaccharide preparation fromExample 4 were prepared and blended into a single lot to produceoligosaccharide 9.4.

Example 9.5: four batches of the oligosaccharide preparation fromExample 5 were prepared and blended into a single lot to produceoligosaccharide 9.5.

Example 9.6: two batches of the oligosaccharide preparation from Example6 were prepared and blended into a single lot to produce oligosaccharide9.6.

Example 9.7: two batches of the oligosaccharide preparation from Example7 were prepared and blended into a single lot to produce oligosaccharide9.7.

Further structural variants of oligosaccharide preparations of Examples1-7 were synthesized at 300 gram scale using the methods of Examples 1-7but varying the starting sugar compositions, acid, acid loading, time,and reaction temperature. Oligosaccharide preparations were synthesizedas follows:

Example 9.8: 300 grams of sucrose, 3 grams of phosphoric acid, and 27grams of water were reacted at 125° C. for about one hour to obtain adark brown oligosaccharide syrup that was then diluted to 60 Brix withdistilled water.

Example 9.9: 270 grams of glucose, 30 grams of sucrose, 0.3 grams ofphenylphosphonic acid, and 27 grams of water were reacted at 130° C. forbetween one to four hours to obtain a dark brown oligosaccharide syrupthat was then diluted to 60 Brix with distilled water.

Example 9.10: 225 grams of glucose, 75 grams of lactose, 3 grams ofbutylphosphonic acid and 27 grams of water were reacted at 130° C. forbetween one to four hours to obtain a dark amber oligosaccharide syrupthat was then diluted to 60 Brix with distilled water.

Example 9.11: 225 grams of glucose, 75 grams of lactose, 3 grams ofphenylphosphonic acid and 27 grams of water were reacted at 130° C. forbetween one to five hours to obtain a dark amber oligosaccharide syrupthat was then diluted to 60 Brix with distilled water.

Example 9.12: 270 grams of glucose, 30 grams of lactose, 3 grams ofphenylphosphinic acid and 27 grams of water were reacted at 130° C. forbetween three to five hours to obtain a dark brown oligosaccharide syrupthat was then diluted to 60 Brix with distilled water.

Example 9.13: 300 grams of glucose, 3 grams of phenylphosphinic acid,and 27 grams of water were reacted at 130° C. for one to three hours toobtain a dark amber oligosaccharide syrup that was then diluted to 60Brix with distilled water.

Example 9.14: 300 grams of glucose, 2 grams of propionic acid, and 27grams of water were reacted at 130° C. for one to four hours to obtainan amber oligosaccharide syrup that was then diluted to 60 Brix withdistilled water.

Example 9.15: 300 grams of glucose, 0.15 grams of8-hydroxy-5-quinolinesulfonic acid hydrate, and 27 grams of water werereacted at 130° C. for two to four hours to obtain an amberoligosaccharide syrup that was then diluted to 60 Brix with distilledwater.

In the above reactions, all masses refer to the pure component masses,and the total mass of reactant water was inclusive of any carry-alongwater provided by the moisture content and/or water of hydration of thereactant sugars.

Characterization of Oligosaccharide Preparations:

The resulting materials were analyzed by HPLC Size ExclusionChromatography (SEC) to characterize the molecular weight distribution,LC-MS/MS analysis to quantify the DP2 anhydrosugar content, and 2D ¹H,¹³C-HSQC NMR to fingerprint the molecular structure of the correspondingoligosaccharide preparations.

Polymer Molecular Weight determined by HPLC:

The number average molecular weight (MWn) and weight-average molecularweight (MWw) of the oligosaccharide preparations of Examples 9.1-9.7were determined by HPLC. SEC analysis was performed on an Agilent 1100series HPLC with refractive index detection using an Agilent PLaquagel-OH 20 column at 40° C. with distilled water at 0.45 mL/min asthe mobile phase. Retention-time to MW calibration was performed usingstandard solutions with known molecular weight and standard methods fromthe art were used to determine the various distribution properties fromthe SEC chromatogram. The MWn and MWw of oligosaccharide preparationswith multiple lots are shown below in Table 1.

TABLE 1 MWn and MWw of Oligosaccharide Preparations OligosaccharidePreparation MWn (g/mol) MWw (g/mol) 9.1 719 ± 11 1,063 ± 23 9.2 808 ± 30 1,336 ± 122 9.3 757 ± 15 1,186 ± 49 9.4 761 1,196 9.5 755 1,177 9.6 505709 9.7 762 ± 12 1,154 ± 14

Anhydro-DP2 Content Analysis by GC-MS:

The anhydro DP2 content of oligosaccharide preparations was determinedby LC-MS/MS using a Capcell Pak NH2 (Shiseido; 250×4. 6 mm, 5 μm) columnat a flowrate of 1 mL/min under isocratic conditions ofwater/acetonitrile 35/65. Prior to MS the flow was split 1:4 and amakeup flow of 50 μL 0.05% NH₄OH was added to enhance ionization. For MSdetection ESI probe was used in negative mode and MRM method allowedtargeted analysis.

The anhydro DP2 contents of the oligosaccharide preparations was firstdetermined relative to that of the oligosaccharide preparation ofExample 9.7 as a reference composition. The absolute anhydro DP2 contentof the reference oligosaccharide preparation of Example 9.7 was thendetermined by HPLC-MS/MS to be about 10% and the anhydro DP2 contents ofthe oligosaccharide preparations of Examples 9.1 to 9.6 were thenobtained by calculation. The relative and absolute DP2 contents weredetermined as described in Table 2.

TABLE 2 Anhydro DP2 content for oligosaccharide preparations withmultiple lots Relative Anhydro Anhydro DP2 Oligosaccharide DP2 Content %Content (g Preparation Relative to Ex 9.7 AHDP2/g total DP2) 9.1 53%5.3% 9.2 14% 1.4% 9.3 57% 5.7% 9.4 53% 5.3% 9.5 33% 3.3% 9.6 50% 5.0%9.7 100%  10.0%

Molecular Fingerprint by 2D ¹H, ¹³C-HSQC NMR:

The molecular structures of the oligosaccharide preparations of Example9 were characterized by 2D ¹H, ¹³C-HSQC NMR spectroscopy. Samples wereprepared by drying 125 mg (dry solids basis) of the oligosaccharidepreparation at 40° C. and re-dissolving in D₂O containing 0.1% acetone.NMR spectra were acquired at 300K on either a Bruker Avance NMRspectrometer operating at a proton frequency of 400 MHz or on a BrukerAvance III NMR spectrometer operating at a proton frequency of 600 MHzequipped with a cryogenically cooled 5 mm TCI probe. FIG. 1 provides anillustrative 2D ¹H,¹³C HSQC NMR spectrum of the oligosaccharidepreparation of oligosaccharide preparation 9.7.

The anomeric region of the ¹H, ¹³C-HSQC spectrum, F2 (δ¹H)=4.2-6.0 ppmand F1(δ¹³C)=90-120 ppm, was used to fingerprint the linkagedistribution of the oligosaccharide preparations. Each peak in theanomeric region was integrated and its relative abundance was determinedrelative to that of the total anomeric region. 2D ¹H,¹³C HSQCfingerprinting was performed on the four lots of the oligosaccharidepreparation 9.1.

TABLE 3 Relative Abundances of F2 and F1 Peaks of OligosaccharidePreparation 9.1 F2 (ppm) F1 (ppm) AUC (Average ± SEM) 5.43 92.42 0.4% ±0.3% 5.44 102.07 0.4% ± 0.1% 5.43 90.05 0.5% ± 0.2% 5.40 100.22 1.6% ±0.4% 5.37 98.33 0.7% ± 0.4% 5.35 99.70 2.7% ± 0.6% 5.33 96.53 0.3% ±0.2% 5.24 100.86 0.5% ± 0.2% 5.22 92.71 20.2% ± 3.9%  5.21 102.45 0.5% ±0.4% 5.18 93.86 0.9% ± 0.4% 5.17 96.01 0.4% ± 0.1% 5.09 96.88 0.6% ±0.3% 5.03 108.49 0.4% ± 0.2% 5.02 109.16 0.4% ± 0.4% 4.98 99.19 0.6% ±0.3% 4.95 98.51 30.6% ± 4.1%  4.86 98.53 0.7% ± 0.5% 4.79 96.84 0.6% ±0.3% 4.71 103.48 2.5% ± 0.7% 4.64 103.56 0.8% ± 0.4% 4.63 102.49 0.7% ±0.5% 4.62 104.56 1.4% ± 0.4% 4.57 97.07 1.6% ± 0.3% 4.50 103.30 25.9% ±2.2%  4.45 103.56 2.4% ± 1.3%

Example 10 Determination of the Anhydro Sugar Subunits of anOligosaccharide Preparation

The relative abundance of anhydro sugar subunits in the oligosaccharidepreparations of Example 9 was determined by MALDI-MS on a BrukerUltraflex instrument. Samples were dissolved in water to a concentrationof 10 mg/ml, from which 5 μl were mixed with matrix solution (30 mg/mlDHB in 80% ethanol and water in a ratio 1:10). Plates were prepared byapplying 1 μl of the analyte solution to the target plate and dried atambient air. In some cases, samples were re-crystalized by applying 1 μlethanol prior to MS analysis.

FIG. 2 provides an illustrative MALDI spectrum of an oligosaccharidepreparation from Example 9. Anhydro-sugar subunits are clearly observedas offset peaks shifted by −18 g/mol relative to its respectiveprincipal DP parent. Offset peaks are observed at all values of DP,indicating that anhydrosugar subunits are detected at alloligosaccharide sizes. The relative intensity of the anhydro subunitpeak was determined to be about 10% of the total peak intensity for eachDP, from which the total anhydro subunit relative abundance wasdetermined to be about 10%. FIG. 25A and FIG. 25B illustrate MALDIspectra of an oligosaccharide preparation from Example 2. Anhydro-sugarsubunits are observed at every DP level with a relative intensity in therange of 5-10%.

Example 11 Characterization of the Anhydro Sub-Units of anOligosaccharide Preparation

The anhydrosugar subunits of the oligosaccharide preparations of Example9 were characterized using a combination of LC-MS, GC-MS, LC-MS/MS, andNMR methods.

Characterization of Anhydro-DP1 Components:

The anhydro DP1 component of an oligosaccharide preparation from Example9 was isolated by preparative liquid chromatography. The isolatedanhydro-DP1 component was prepared for NMR by dissolving it in 0.75 mLof D₂O. FIG. 3 provides an illustrative 1D ¹H-NMR spectrum of an anhydroDP1 fraction isolated from an oligosaccharide of Example 9 and FIG. 4provides an illustrative APT ¹³C-NMR spectrum of the same isolatedanhydro DP1 fraction.

Using the following peak assignments in Table 4, the ratio of1,6-anhydro-beta-D-glucofuranose to 1,6-Anhydro-beta-D-glucopyranose wasdetermined by NMR to be 2:1.

TABLE 4 NMR Peak Assignments 1,6-anhydro-beta- 1,6-Anhydro-beta-D-glucofuranose D-glucopyranose # ¹H (ppm) ¹³C (ppm) ¹H (ppm) ¹³C (ppm)1 5.33 101.9 5.01 104.4 2 3.40 70.6 4.37 79.8 3 3.56 (ov)^(a) 73.0 4.2778.3 4 3.56 (ov)^(a) 71.3 4.38 80.6 5 4.50 76.7 3.74 64.1 6 3.97, 3.6465.7 4.14, 3.72 66.7 ^(a)Ov indicates overlapped signal

Characterization of the Anhydro-DP2 Components

The anhydrosugar subunits of the oligosaccharide preparations of Example9 were characterized using a combination of LC-MS, GC-MS, LC-MS/MS, andNMR methods. The anhydro DP2 content of the oligosaccharide preparationsof Example 9 were determined by GC-MS and LC-MS/MS analysis. Gaschromatography was performed using a 30 m×0.25 mm fused silica columncontaining HP-5MS stationary phase, with 21.57 psi constant pressureHelium as the carrier gas. Aliquots were pre-derivatized by acetylationby dissolving 20 mg of sample in 0.5 mL pyridine with 0.5 mL aceticanhydride for 30 minutes at 60° C. 1 uL samples were injected at 300° C.with an oven temperature program starting at 70° C. and ramping by 10°C. per minute to 315° C. Detection was performed on an Agilent 5975C MSDwith an electron energy of 70 eV.

FIG. 5 illustrates an enlargement of the GC-MS chromatogram for theoligosaccharide preparation 9.7. The TIC and XIC (m/z 229) plotsdemonstrate that the DP2 and anhydro-DP2 components are clearlyresolved. FIGS. 30A-30B, 31A-31B, 32A-32B, and 33A-33B illustrate thepresence of the DP1, anhydro DP1, DP2 and anhydro DP2 fractions asdetected by GC-MS in an oligosaccharide preparation of Example 1,Example 3, Example 4, and Example 7, respectively. As shown in FIGS.30A-30B, 31A-31B, 32A-32B, and 33A-33B, anhydro DP1 and DP1 fractionshave a retention time of from about 12-17 minutes, and anhydro DP2 andDP2 fractions have a retention time of about from 22-25 minutes.

FIG. 36 illustrates MALDI-MS spectra comparing the oligosaccharidepreparation from Example 9 at different laser energies. Relativeabundancy of signals was nearly unchanged, demonstrating that no loss ofwater is introduced by the laser ionization. Hence, proving the presenceof anhydro-sugar subunits in the oligosaccharide preparation.

Example 12 Observation of Caramelization Subunits in an OligosaccharidePreparation

An oligosaccharide preparation comprising a 5-hydroxymethylfurfuralcaramelization subunit was demonstrated by a combination of HPLCanalysis and 2D ¹H,¹³C HSQC NMR. A 50 mg aliquot of an oligosaccharidepreparation from Example 9 was dissolved in 0.8 mL D₂O. The resulting 2D¹H, ¹³C HSQC spectrum was analyzed for the presence of a glycosidiclinkage between 5-hmf and the anomeric carbon of glucose, with thefollowing peak assignments: ¹H NMR: δ=9.39 ppm (CHO, m), 7.44 ppm (Ar—H,m), 6.68 ppm (Ar—H, m), 4.60 ppm (CH2, m); and ¹³C NMR: δ=180.0, 150.6,126.2, 112.7, 159.9, 64.5. The absence of free 5-hmf was confirmed byHPLC using an Agilent 1100 HPLC system equipped with a 3000 mm Bio-RadAminex 87H using 25 mM aqueous sulfuric acid at 0.65 mL/min isocraticelution and UV detection. 5-hmf was quantified against an authenticsample. No 5-hmf peak was observed in the oligosaccharide preparation ofExample 9, but was observed following low-temperature acid hydrolysis ofthe oligosaccharide preparation under conditions not expected to producefree 5-hmf in the hydrolysate. FIG. 6 provides an illustrative 2D HSQCspectrum of an oligosaccharide preparation that incorporates acaramelization subunit.

Example 13 Comparative Example

A commercial 5 kDa dextran was analyzed by MALDI-MS for the presence ofanhydrosugar subunits. FIG. 7 shows the clear presence of the offsetpeak shifted −18 g/mol from the principal DP peak (Na+ adduct at 851.268g/mol). By contrast the dextran sample was found to be essentially freeof anhydro sugar subunits.

Example 14 Quantification of the Anhydro DP Component by LC-MS/MS

The DP2 fraction of oligosaccharide preparations was isolated by liquidchromatography. Samples were dissolved in water and separated using aCapcell Pak NH2 (Shiseido; 250×4. 6 mm, 5 μm) column at a flowrate of 1mL/min under isocratic conditions of water/acetonitrile 35/65. In somecases, following chromatographic separation, 50 μL 0.05% NH₄OH was addedto enhance ionization. The anhydro DP2 content was determined by MS/MSdetection. For MS detection ESI probe was used in negative mode and MRMmethod allowed targeted analysis. FIG. 8A illustrates detection anoligosaccharide preparation from Example 9 over a concentration range of1-80 μg/mL in water, with a linear calibration curve (shown in FIG. 8B)from the area under the LC-MS/MS chromatogram to concentration.

FIGS. 26A-26C, 27A-27C, 28A-28C, and 29A-29C illustrate the presence ofthe anhydro DP2, anhydro DP1, and DP2 species detected by LC-MS/MS in anoligosaccharide preparation of Example 1, Example 3, Example 4, andExample 7, respectively.

Example 15 Preparation of Feed Comprising Oligosaccharide Preparations

Poultry and swine diets were prepared to demonstrate the incorporationof oligosaccharide preparations into the diet. Control feeds exhibitinga variety of ingredient compositions and corresponding treated feedsobtained by augmenting the respective control feeds with theoligosaccharide preparations of Example 9 were prepared as follows:

Example Feed 15.1: Control Feed 15.1 (CTR) was prepared using 62% cornmeal and 32% soybean meal. Treated Feed 15.1 (TRT) was prepared byaugmenting the control feed 15.1 (CTR) with 500 mg/kg of anoligosaccharide preparation from Example 9. For the treated diet, theoligosaccharide preparation was provided in a powder form by drying theoligosaccharide onto ground rice hulls as a carrier and adding thepowder to the mixer using a micro-ingredient balance prior to pelleting.

Example Feed 15.2: Control Feed 15.2 (CTR) was prepared using 62% cornmeal, 3% soybean concentrate, and 26% soybean meal. Treated Feed 15.2(TRT) was prepared by augmenting the control feed 15.2 (CTR) with 500mg/kg of an oligosaccharide preparation from Example 9. For the treateddiet, the oligosaccharide preparation was provided in a powder form bydrying the oligosaccharide onto ground rice hulls as a carrier andadding the powder to the mixer using a micro-ingredient balance prior topelleting.

Example Feed 15.3: Control Feed 15.3 (CTR) was prepared using 52% cornmeal, 6% corn starch, 5% soybean concentrate, 26% soybean meal, and atitanium oxide micro-tracer. Treated Feed 15.3 (TRT) was prepared byaugmenting the control feed 15.3 (CTR) with 500 mg/kg of anoligosaccharide preparation. For the treated diet, the oligosaccharidepreparation was provided in a powder form by drying the oligosaccharideonto ground rice hulls as a carrier and adding the powder to the mixerusing a micro-ingredient balance prior to pelleting.

Example Feed 15.4: Control Feed 15.4 (CTR) was prepared using 55% cornmeal and 39% soybean meal. Treated Feed 15.4 (TRT) was prepared byaugmenting the control feed 15.4 (CTR) with 1,000 mg/kg of anoligosaccharide preparation. For the treated diet, the oligosaccharidepreparation was provided in a powder form by drying the oligosaccharideonto ground rice hulls as a carrier and adding the powder to the mixerusing a micro-ingredient balance prior to pelleting.

Example Feed 15.5: Control Feed 15.5 (CTR) was prepared using 62% cornmeal, 3% soybean concentrate, and 26% soybean meal. Treated Feed 15.5(TRT) was prepared by augmenting the control feed 15.5 (CTR) with 500mg/kg of an oligosaccharide preparation from Example 9. For the treateddiet, the oligosaccharide preparation was provided in a powder form andadding the powder to the mixer using a micro-ingredient balance prior topelleting.

Example Feed 15.6: Control Feed 15.6 (CTR) was a commercial U.S.corn-soy starter poultry feed. Treated Feed 15.6 (TRT) was a commercialU.S. corn-soy starter poultry feed containing 500 ppm of anoligosaccharide preparation. For the treated diet, the oligosaccharidepreparation was provided in a powder form and adding the powder to themixer using a micro-ingredient balance prior to pelleting.

Example Feed 15.7: Control Feed 15.7 (CTR) was a research corn-soypoultry feed with the following diet composition: corn meal 62.39%,soybean meal 31.80%, calcium carbonate 0.15%, bicalcic-phosphate 2.2%,sodium chloride 0.15%, DL-Methionine 0.15%, L-Lysine 0.10%, Soya Oil2.00%, vitamin-mineral premix 1.00%, and coccidiostat 0.06%. TreatedFeed 15.7 (TRT) was obtained by supplementing the control feed 15.7(CTR) with 1000 ppm of the oligosaccharide preparation of Example 9.1.For the treated diet, the oligosaccharide preparation was provided as60% syrup in water and was applied by spraying the syrup onto the feedpost pelleting.

Example Feed 15.8: Control Feed 15.8 (CTR) was a research corn-soypoultry feed with the following composition: wheat meal 48.45%, soybeanmeal 32.00%, rye 12%, calcium carbonate 0.20%, bicalcic phosphate 2.00%,sodium chloride 0.20%, DL-methionine 0.15%, soya oil 4.00%,vitamin-mineral premix 1.00%. Treated Feed 15.7 (TRT) was obtained bysupplementing the control feed 15.7 (CTR) with 1000 ppm of theoligosaccharide preparation of Example 9.3. For the treated diet, theoligosaccharide preparation was provided as 60% syrup in water and wasapplied by spraying the syrup onto the feed post pelleting.

As would be understood by one skilled in the art, the 15.1-15.6 alsocontained industry standard levels of fat, vitamins, minerals, aminoacids, other micronutrients and feed enzymes. In some cases the feedscontained a cocciodiostat, but were free in all cases of antibioticgrowth promoters. The feeds were provided as either mash, pelletized, orcrumbled diets, according to standard practices.

Example 16 Extraction of Feed Samples

Diets prepared according to the procedures of Example 15 were processedby extraction. Feed samples were ground with a mill. Five grams of theresulting milled feed were weighted into a 50 mL volumetric flask andhot water (approx. 80° C.) was added. After shaking, the mixture wasincubated in an ultrasonic water bath at 80° C. for 30 minutes. Aftercooling, the solution was centrifuged for 20 min at 3000×g and thesupernatant was filtered through a 1.2 μm filter followed by a 0.45 μmfilter (and in some cases by a 0.22 μm filter). The resulting filteredsolutions were evaporated to dryness with a rotary evaporator.

In some cases, the extraction was performed using 50 wt % ethanol inwater as an alternative extraction solvent. In some cases, thefiltration step was performed using a 5,000 Dalton molecular-weightcutoff membrane filter.

Example 17 Enzymatic Processing of Feed Extracts

The feed extracts of Example 16 were subjected to one or more enzymatichydrolysis steps to digest oligosaccharides naturally present in thefeed. A mixture of α-Amylase and amyloglycosidases were used to digestα(1,4) linkages of gluco-oligosaccharides and starch. Invertase andα-galactosidase were used to remove sucrose, raffinose, and other commonfiber saccharides.

Enzyme solutions were prepared as follows: Amyloglucosidase (A. niger)36000 U/g solution at 800 U/mL in ammonium acetate buffer (ammoniumacetate 0.2 M pH 5 containing 0.5 mM MgCl2 and 200 mM CaCl2), α-Amylase(Porcine Pancreas) 100000 U/g Megazyme: solution at 800 U/mL in ammoniumacetate, Invertase from Backer's yeast (S. cerevisiae) 300 U/mg Sigma:solution at 600 U/mL in ammonium acetate buffer, α-Galactosidase from A.niger Megazyme 1000 U/mL.

The dry feed extracts of Example 16 were re-suspended in 10 mL ammoniumacetate buffer. 50 μl of α-amylase (4 U/mL final), 50 μl ofamyloglucosidase (4 U/mL final), 50 μl of invertase (3 U/mL final) wereadded. 20 μl α-galactosidase (2 U/mL final) was added optionally. Thesolution was incubated for 4 hours at 60° C. The digested extract wasthen filtered through an ultrafiltration filter (Vivaspin Turbo 4, 5000MWCO, Sartorius) before being evaporated to dryness on a nitrogenevaporation system. In variations of the enzymatic digestion, one ormore of the above enzymes were used in combinations and the digestionperiod was varied between 4 hours to overnight digestion. The enzymeconcentrations were varied up to twice the above loadings, and the fullenzymatic digestion procedure was repeated multiple times in sequence onthe same feed.

TABLE 5 List of Feed Samples for Extraction and Digestion ExtractionMatrix solvent Filtration Enzyme digestion 1 Blank feed Water 0.22 μM —2 Anhydro-Oligomers feed 1000 mg/kg Water 0.22 μM — 3 Blank Feedethanol/water 0.22 μM — 50/50 4 Anhydro-Oligomers feed 1000 mg/kgethanol/water 0.22 μM — 50/50 5 Anhydro-Oligomers Water 0.22 μM a + b (4h 60° C.) 6 Blank feed Water 0.22 μM a + b (4 h 60° C.) 7Anhydro-Oligomers feed 1000 mg/kg Water 0.22 μM a + b (4 h 60° C.) 10Blank feed ethanol/water 0.22 μM — 50/50 11 Anhydro-Oligomers feed 1000mg/kg ethanol/water 0.22 μM — 50/50 12 Anhydro-Oligomers feed 1000 mg/kgWater 0.45 μM a + b (4 h 60° C.) 13 Anhydro-Oligomers feed 1000 mg/kgWater 0.22 μM a + b (4 h 60° C.) 14 Blank starter feed A Water 0.45 μMa + b (4 h 60° C.) 15 Anhydro-Oligomers starter feed B 2000 Water 0.45μM a + b (4 h 60° C.) mg/kg 16 Blank feed Water 0.45 μM a + b + c (4 h60° C.) 17 Anhydro-Oligomers feed 1000 mg/kg Water 0.45 μM a + b + c (4h 60° C.) 18 Rice spelt/Anhydro-Oligomers Water 0.45 μM — 19 Ricespelt/Anhydro-Oligomers ethanol/water 0.45 μM — 50/50 20 Blank feedWater 0.45 μM a + b + c (4 h 60° C.) 21 Anhydro-Oligomers feed 1000mg/kg Water 0.45 μM a + b + c (4 h 60° C.) 22 Grower feed C (blank)Water 0.45 μM 23 Grower feed D (Anhydro-Oligomers Water 0.45 μM 2000mg/kg) 24 Pre starter feed A (blank) Water 0.45 μM 25 Pre starter feed D(Anhydro-Oligomers Water 0.45 μM 1000 mg/kg) 26 Grower feed C (blank)Water 0.45 μM a + b + c + d (4 h 60° C.) 27 Grower feed D(Anhydro-Oligomers Water 0.45 μM a + b + c + d (4 h 2000 mg/kg) 60° C.)28 Pre starter feed A (blank) Water 0.45 μM a + b + c + d (4 h 60° C.)29 Pre starter feed D (Anhydro-Oligomers Water 0.45 μM a + b + c + d (4h 1000 mg/kg) 60° C.) 30 Maize Water 0.45 μM — 31 Wheat Water 0.45 μM —32 Soy Water 0.45 μM — 33 Maize Water 0.45 μM a + b + c + d (4 h 60° C.)34 Wheat Water 0.45 μM a + b + c + d (4 h 60° C.) 35 Soy Water 0.45 μMa + b + c + d (4 h 60° C.) 41 Blank Feed Water 0.45 μM 42Anhydro-Oligomers feed 1000 mg/kg Water 0.45 μM 43 Blank feed Waterultra 5000 a + b + c X2 MWCO (overnight 60° C.) 44 Anhydro-Oligomersfeed 1000 mg/kg Water ultra 5000 a + b + c X2 MWCO (overnight 60° C.)Anhydro-Oligomers refer to anhydro-subunit containing oligosaccharides.Blank feeds refer to nutritional compositions without addedanhydro-oligomers.Enzyme a=Amyloglucosidase (A. niger) 36000 U/g MegazymeEnzyme b=α-Amylase (Porcine Pancreas) 100000 U/g MegazymeEnzyme c=Invertase from Baker's yeast (S. cerevisiae) 300 U/mg SigmaEnzyme d=α-Galactosidase from A. niger 620 U/mg Megazyme

Example 18 Detection of Oligosaccharide Preparations in Feed

The Control and Treated diets according to Example 15 were assayed todetect the absence or presence of oligosaccharide preparations. Afterdiet manufacture, 1 kg samples were drawn from the final feed. Theextractable solids content of the feed was obtained by water extractionusing the procedure of Example 16. The resulting extracts were analyzedfor the presence of anhydro-DP species by LC-MS/MS according to theprocedure of Example 14.

FIG. 9 shows the absence of anhydro-DP2 species in the control feeds ofExamples 15.1(CTR)-15.6(CTR) versus the presence of anhydro-DP2 speciesin the treated feeds of Examples 15.1(TRT)-15.6(TRT). Integration of theresulting LC-MS/MS chromatograms was used to determine the presence ofthe oligosaccharide preparations of Example 9 in the final feed.Specifically, feeds were determined to contain the respectiveoligosaccharide preparation if the area under the anhydro-DP2 peakexceeded the limit of detection (or any other suitable thresholdestablished in the method).

Example 19 Quantification of Oligosaccharide Preparations in Feed

The Control and Treated diets according to Example 15 were assayed todetermine the concentration of oligosaccharide preparations in the finalfeed. After diet manufacture, 1 kg samples were drawn from the finalfeed. The extractable solids content of the feed was obtained by waterextraction using the procedure of Example 16. The resulting extractswere analyzed for the presence of anhydro-DP species by LC-MS/MSaccording to the procedure of Example 14 and the area of the anhydro-DP2peak was compared against a standard calibration curve to determine theconcentration of the oligosaccharide preparation in the feed (Table 6).

TABLE 6 Concentration of Oligosaccharide Preparation in FeedOligosaccharide Content Oligosaccharide Content Feed (ppm) in ControlFeed (ppm) in Treated Feed Example 15.1 Not detected 1642 Example 15.2Not detected 953 Example 15.3 Not detected 1912 Example 15.4 Notdetected 549 Example 15.5 Not detected 406 Example 15.6 Trace 401

Example 20 NMR Characterization of Anhydro-Subunit ContainingGluco-oligosaccharides

Gluco-oligosaccharide preparations comprising anhydro-subunits werecharacterized by i) the degree of polymerization and ii) the glycosidiclinkage distribution of the glucose units.

The relative molar abundances of α(1,1)α, α(1,1)β, β(1,1)β, α(1,2),β(1,2), α(1,3), β(1,3), α(1,4), β(1,4), α(1,6), and β(1,6) linkages wereidentified by NMR spectroscopy. ¹H NMR chemical shifts for the anomericprotons were determined as follows: the region d=4.5-5.5 ppm wasconsidered, with the resonances of C-2 to C-6 covalently bound clusteredat ˜d 3.2 and 3.9 ppm. Due to coupling with the H-atom at C-2, theanomeric proton appears as a doublet and the axial position appears athigher field than the equatorial position. The sugar conformation waselucidated from the coupling constant of neighboring protons:equatorial-equatorial, equatorial-axial (small coupling constants) oraxial-axial (larger coupling constants).

Elucidation of glycosidic linkages was also performed by ¹³C NMR.Primary, secondary, tertiary, and quaternary carbons were distinguishedby proton off-resonance decoupling or polarization transfer. Carbonsattached to methoxy groups resonate at a lower field than correspondingcarbon atoms with free hydroxy groups and ring carbon atoms with axialhydroxy groups generally absorb at higher field than correspondingcarbons with equatorial hydroxy groups. Thus, following theseguidelines, and comparing with reported chemical shifts for both ¹H and¹³C of analogous sugars in literature most of the signals were assigned.

For the determination of the linkage distribution, J-RES and ¹H,¹³C-HSQC were used. For some samples, the HSQC method showed a superiorperformance. For each analysis, approximately 50 mg of freeze-driedproduct were dissolved in D2O and transferred to a 5 mm NMR tube. Anyresidual catalyst or solids were removed by filtration. NMR experimentswere performed on a Bruker Avance III NMR spectrometer operating at 600MHz proton corresponding to 150 MHz carbon Larmor frequency. Theinstrument was equipped with a cryogenically cooled 5 mm TCI probe. Allexperiments are carried out at 298 K. ¹H NMR spectra were recorded andcalibrated in deuterated water (4.75 ppm). ¹³C NMR spectra arecalibrated with acetone (30.9 ppm). Data were acquired using TopSpin 3.5and processed with ACD/Labs running on a personal computer.

FIG. 10 provides a representative 2D-1H JRES NMR spectrum of ananhydro-subunit containing gluco-oligosaccharide sample with solventpre-saturation. Assignments of the different glycosidic linkages weremade according to Table 7.

TABLE 7 Relative Molar Abundance for Glycosidic Linkages in anAnhydro-subunit Containing Gluco-oligosaccharides Sample (2D-1H JRES NMRMethod) Relative Molar Abundance Relative Molar Abundance Linkage Testedin Lab I Tested in Lab II α(1, 2) 10.1% 9.2% α(1, 4) 2.0% 17.0% α(1, 3)4.5% 1.3% α(1, 6) 28.9% 33.6% β(1, 2) 5.7% 6.5% β(1, 3) 13.3% 6.3% β(1,4) 17.9% 10.7% β(1, 6) 18.9% 14.5%

As shown in Table 7, for some samples, discrepancies were observedbetween experiments performed by different labs using differentinstruments for the 2D-¹H JRES NMR analysis.

FIG. 11 provides a representative ¹H, ¹³C-HSQC NMR spectrum of ananhydro-subunit containing gluco-oligosaccharides sample with relevantresonances and assignments used for linkage distribution. By contrastthe determination by ¹H, ¹³C-HSQC NMR was found to be consistent betweentwo different labs and instruments, as shown in Table 8.

TABLE 8 Relative Molar Abundance for Glycosidic Linkages in FourAnhydro-subunit Containing Gluco-oligosaccharides Samples (¹H, ¹³C- HSQCNMR Method) Sample 1 Sample 2 Sample 3 Sample 4 Linkage Lab I Lab II LabI Lab II Lab I Lab II Lab I Lab II α(1, 2) 9.2% 9.2% 9.0% 9.5% 9.1% 9.9%9.1% — α(1, 4) 1.4% 1.3% 1.2% 1.3% 1.3% 1.3% 0.0% — α(1, 3) 17.7% 17.0% 17% 17.7% 17.5% 16.7% 21.9% — α(1, 6) 33.9% 33.6% 33.6%  30.9% 36.0%31.6% 34.4% — β(1, 2) 5.7% 6.5% 5.7% 7.6% 5.2% 7.6% 5.2% — β(1, 3) 4.1%6.3% 4.2% 6.2% 4.4% 6.1% 5.6% — β(1, 4) 8.5% 10.7% 8.9% 10.7% 7.6% 10.7%8.3% — β(1, 6) 12.3% 14.5% 12.6%  11.6% 11.7% 11.6% 11.0% —

Diffusion-Ordered NMR Spectroscopy (DOSY) was performed to separate theNMR signals of different species according to diffusion coefficient andthus MW. Signals at the upper part of the DOSY spectra in FIG. 12correspond to high DP species, while lower DPs species appear below.FIG. 12 illustrates an overlay of ¹H DOSY spectra of threeanhydro-subunit containing oligosaccharides in Table 8.

Example 21 Semi-Preparative Isolation of DP1 & DP2 Fraction

Preparative isolation of the DP1 fraction was performed by preparativeHPLC using a Waters BEH Amide 19×150 mm column. As mobile phase waterwas used as solvent A and Acetonitrile as solvent B, each with 0.1%ammonia. The applied gradient is shown in Table 9. The collected DP1fraction of 8 separations were pooled, dried and resolubilized in 0.75ml D₂O for NMR analysis as described before.

For the characterization of the DP2 fraction a 2-step purification wasdone. The first step was performed on a flash chromatography system,using an ELSD (Evaporative light scattering detector). 2 ml (2.65 g) ofan oligosaccharide preparation were diluted with 1 ml DMSO, 0.5 ml waterand 0.5 ml Acetonitrile. The solution was mixed and for 15 minsonicated. 1 ml of the solution was injected on a YMC DispoPackAT, NH2,spherical, 25 μm, 120 g column was used. The oligosaccharide preparationwas separated running an isocratic gradient method with 75% Actonitrilein water at 40 ml/min. The DP2 containing fraction was dried withnitrogen and resolubilized in DMSO/water (80:20, v/v).

For the 2^(nd) purification step an analytical UPLC system with a YMCNH₂ 4.6×250 mm (5 μm) column at 40° was used. The DP2 fraction waspurified with an isocratic gradient (Table 10) and a flow rate of 1ml/min. A 1:5 post-column spilt was used in order to trigger thecollection by ELSD. The DP2 fraction from 12 chromatographic runs werepooled, the Acetonitrile removed by heated nitrogen and the residualwater by freeze-drying. The dry fraction was resolubilized forsubsequent LC-MS/MS & NMR analysis.

TABLE 9 Gradient Method Time (min) Flow rate (ml/min) Solvent A SolventB 0 25 10 90 2.5 25 10 90 23 25 25 75 23.1 25 10 90 47 25 10 90

TABLE 10 Isocratic Method Time (min) Flow rate (ml/min) Water (%) ACN(%) 0 1 25 75 15 1 25 75

Example 22 Synthesis of an Oligosaccharide Preparation with aMonotonically Decreasing DP Distribution

330 grams of D-glucose monohydrate and 0.3 grams of(+)-Camphor-10-sulfonic acid were added to a one-liter, three-neck flaskwith overhead mechanical mixing provided by high-torque mechanical mixerthrough a flex coupling. The flask was secured inside a hemisphericalelectric heating mantle operated by a temperature control unit via awand thermocouple inserted into the reaction mixture. The tip of thethermocouple was adjusted to reside within the reaction mixture withseveral mm clearance above the mixing element. The flask was equippedwith a reflux condenser cooled by a water-glycol mixture maintainedbelow 4° C. by a recirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. When the reaction temperature wasincreased to between 120° C. and 130° C., the apparatus was switchedfrom a reflux condenser to a distillation configuration with a roundbottom receiving flask placed in an ice bath. The reaction wasmaintained at 130° C. with 120 RPM mixing for sixty minutes and the massof condensate collected in the receiving flask was recorded as afunction time at 10 minute intervals. The reaction was quenched byadding distilled water and removing the heat. After the product mixturecooled to room temperature, an aliquot of the product syrup was dilutedto about 1 Brix as determined by refractive index. The diluted aliquotwas microfiltered using a 0.2 micron syringe filter and analyzed by HPLCsize exclusion chromatography (SEC). SEC analysis was performed on anAgilent 1100 series HPLC with refractive index detection using anAgilent PL aquagel-OH 20 column at 40° C. with distilled water at 0.45mL/min as the mobile phase. Retention-time to MW calibration wasperformed using standard solutions with known molecular weight. The DPequilibrium constant was determined to be K=3.3 and the DP distributionwas found to be monotonically decreasing. FIG. 15 and FIG. 16 show theshape of the DP distribution of different oligosaccharide preparationsof Example 9 as determined by HPLC-SEC.

Example 23 Synthesis of an Oligosaccharide Preparation with aNon-Monotonic DP Distribution

330 grams of D-glucose monohydrate and 0.3 grams of(+)-Camphor-10-sulfonic acid were added to a one-liter, three-neck flaskwith overhead mechanical mixing provided by high-torque mechanical mixerthrough a flex coupling. The flask was secured inside a hemisphericalelectric heating mantle operated by a temperature control unit via awand thermocouple inserted into the reaction mixture. The tip of thethermocouple was adjusted to reside within the reaction mixture withseveral mm clearance above the mixing element. The flask was equippedwith a reflux condenser cooled by a water-glycol mixture maintainedbelow 4° C. by a recirculating bath chiller.

The reaction mixture was gradually heated to 135° C. with continuousmixing with a stir rate of 80-100 rpm. When the reaction temperature wasincreased to 130° C., the apparatus was switched from a reflux condenserto a distillation configuration with a round bottom receiving flaskplaced in an ice bath. The reaction was maintained at 135° C. with 120RPM mixing for thirty-five minutes. The reaction was quenched by addingdistilled water and removing the heat. After the product mixture cooledto room temperature, an aliquot of the product syrup was diluted toabout 1 Brix as determined by refractive index. The diluted aliquot wasmicrofiltered using a 0.2 micron syringe filter and analyzed by HPLCsize exclusion chromatography (SEC). SEC analysis was performed on anAgilent 1100 series HPLC with refractive index detection using anAgilent PL aquagel-OH 20 column at 40° C. with distilled water at 0.45mL/min as the mobile phase. Retention-time to MW calibration wasperformed using standard solutions with known molecular weight. The DPdistribution was found to be non-monotonically decreasing. FIG. 16illustrates that the DP3 content is greater than the DP2 content andthat the DP4 and DP5 contents are essentially equal.

Example 24 Fed-Batch Synthesis of an Oligosaccharide Preparation

330 grams of D-glucose monohydrate and 0.3 grams of 2-Pyridinesulfonicacid were added to a one-liter, three-neck flask with overheadmechanical mixing provided by high-torque mechanical mixer through aflex coupling. The flask was secured inside a hemispherical electricheating mantle operated by a temperature control unit via a wandthermocouple inserted into the reaction mixture. The tip of thethermocouple was adjusted to reside within the reaction mixture withseveral mm clearance above the mixing element. The flask was equippedwith a reflux condenser cooled by a water-glycol mixture maintainedbelow 4° C. by a recirculating bath chiller.

The reaction mixture was gradually heated to 130° C. with continuousmixing with a stir rate of 80-100 rpm. When the reaction temperature wasincreased to between 120° C. and 130° C., the apparatus was switchedfrom a reflux condenser to a distillation configuration with a roundbottom receiving flask placed in an ice bath. The reaction wasmaintained at 130° C. with 120 RPM and the mass of condensate collectedin the receiving flask was recorded as a function time at 20 minuteintervals. After 210 minutes, an additional 110 grams of D-glucosemonohydrate were added to the reaction. After 420 minutes, the reactionwas quenched by adding distilled water and removing the heat. After theproduct mixture cooled to room temperature, an aliquot of the productsyrup was diluted to about 1 Brix as determined by refractive index. Thediluted aliquot was microfiltered using a 0.2 micron syringe filter andanalyzed by HPLC size exclusion chromatography (SEC). SEC analysis wasperformed on an Agilent 1100 series HPLC with refractive index detectionusing an Agilent PL aquagel-OH 20 column at 40° C. with distilled waterat 0.45 mL/min as the mobile phase. Retention-time to MW calibration wasperformed using standard solutions with known molecular weight. The DPequilibrium constant was determined to be K=0.8 and the DP distributionwas found to be monotonically decreasing.

Example 25 Growth Performance of Commercial Broiler Chickens Fed anOligosaccharide Preparation

Broiler chickens were grown on the diets of Example 15.6 to determinethe effect of the oligosaccharide preparation on the growth performanceof the animals. Specifically, commercial corn-soymeal poultry feedscontaining dried distillers grains with solubles (DDGS), a coccidiostat,and a standard micronutrient blend, were manufactured according toindustry practices and a three phase feeding program. By proximateanalysis, the feed compositions were determined as shown in Table 11.

TABLE 11 Feed Comnositions With- Component Starter Grower drawal MethodMoisture 13.0% 13.0% 12.9% AOAC 930.15 (drafted oven) Crude 24.1% 21.5%19.6% AOAC 992.15; AOAC 990.03 Protein (CP) Fat (EE) 3.2% 3.8% 3.9% AOAC920.39 (ether extraction) Crude 2.7% 2.4% 2.4% AOAC 962.09 (hydrolysis)Fiber (CF) Ash (AR) 5.2% 4.3% 4.3% AOAC 942.05 (muffle furnace) NFE, by51.9% 55.1% 56.9% Calculated: 1-CP-EE-CF-AR difference Total 100.0%100.0% 100.0%

Control (CTR) and treated (TRT) diets were prepared for each phase asdescribed in Example 15.6, where the treat diets were prepared byaugmenting the control diet with one pound per treated short ton usingthe oligosaccharide preparation of Example 9.7. In total, about 50 shorttons of each diet were manufactured.

Day-of-hatch Hubbard M99×Cobb 500 straight run chicks were obtained froma commercial poultry hatchery and placed randomly into 36 ft×40 ft pensconstructed into a tunnel-ventilated, dirt-floor poultry house.Approximately 30,000 birds were placed in total, with an equal number ofbirds in each pen. The house bedding consisted of built-up littertop-dressed with fresh wood shavings. A standard commercialenvironmental and lighting program were employed. Animals and housingfacilities were inspected daily, including recording the general healthstatus, feed consumption, water supply and temperature of the facility.Any mortalities were recorded daily.

Birds in odd numbered pens were fed the treated diet (i.e., containingthe feed additive at 2 lbs/ton inclusion), and birds in the evennumbered pens received the control diet. All diets were provided adlibitum via automatic feeders in each pen, and on feeder trays from dayone until day 7. Water was provided ad libitum from a nipple drinkingline.

The starter phase took place from day 0 to day 13, the grower phase fromday 14 to day 27, and the withdrawal phase from day 28 through the endof the study, day 31. Bird weights by pen were recorded on days 0 and31. The total mass of consumed feed was recorded for each pen. Weightgain and FCR were then determined for each pen according to standardindustry practices.

On day 31, six male birds were randomly selected from each pen for bloodand cecal sampling. The live weight of each sampled bird was recorded. Ablood sample was collected via wing puncture into vacutainer tubes andfrozen following coagulation and serum separation. Each sampled bird wasthen euthanized via cervical dislocation followed by extraction of thececa using standard veterinary methods. Following dissection, cecalcontents were transferred to 15 mL conical tubes, the weight of thececal contents was recorded, and the contents were flash frozen to −80°C.

From the weights of the sampled birds, the treatment group exhibited an11 point increase in body weight, significant at P<0.05 (by ANOVA).

Example 26 Shotgun Sequencing of Poultry Cecal Microbiota

The relative abundances of identified taxa were determined for a totalof 96 sampled birds obtained from the study of Example 25. For eachmicrobiota sample obtained in Example 25, the cecal contents were thawedand DNA was extracted using standard methods. The extracted DNA wasanalyzed on an Illumina HiSeq-X instrument, with 2×150 bp reads.Standard analyses were performed to process the raw sequencing data,including: trimming (adapter, BBDuk), Entropy filtering (k=5, window=20,min=50, BBDuk), Quality filtering (mean Q20, BBDuk), Gallus filtering(Bowtie2). Taxonomic assignments were made against the MetaPhlAn2(db_v20) database.

Example 27 Microbiota Associated with Improved Growth Performance

To assess the phenotypic connection between microbiota composition andgrowth performance, the sampled birds of Example 25 were grouped intocohorts according to their body weight and feed efficiency. A contrastanalysis was performed to identify microbiota species that differedsignificantly across the various cohorts.

Body Weight Effects:

First a correlation analysis was performed by regressing taxonomicabundance data versus the body weight (BW) of the bird from which thesequencing data were obtained. The correlation and P-Value of thecorrelation were assessed by linear regression to identify candidategenera and species positively correlated with growth performance asmeasured by body weight. Of the approximately 130 species isolated,fewer than thirty exhibited a positive BW correlation (FIG. 17).

The samples birds were grouped into two cohorts: (1) Low BW: the bottomquartile of sampled birds as measured by body weight; and (2) High BW:the top quartile of sampled birds as measured by body weight. Thetaxonomic data from Example 26 were then compared to identify specieswith a statically-significant difference relative abundance between thetwo cohorts. Specifically, for each OTU, the relative abundance of thecorresponding organism was compared between the two cohorts. Specieswere identified which exhibited a higher relative abundance in high BWbirds with P<0.05 as determined by ANOVA. Cecal microbiota associatedwith improved BW performance were characterized by an increased relativeabundance of members of genera Bacteroides, Odoribacter, Oscillibacter,Subdoligranulum, Ruminococcus, and Biophila. The species Odoribactersplanchnicus was found to exhibit a particularly strong correlation withBW. FIG. 17 provides box and scatter plots illustrating four taxapositively associated with BW performance (P<0.05).

Feed Efficiency Effects:

First, a correlation analysis was performed by regressing taxonomicabundance data versus the feed conversion ratio (FCR) of the pencorresponding to the bird from which the sequencing data were obtained.The correlation and P-Value of the correlation were assessed by linearregression to identify candidate genera and species positivelycorrelated with growth performance as measured by feed efficiency. Ofthe approximately 130 species isolated, fewer than twenty exhibited apositive FCR correlation.

The samples birds were grouped into two cohorts: (1) High FCR (i.e.,poor feed efficiency): the top quartile of sampled birds as measured byfeed conversion ratio; and (2) Low FCR (i.e., improved feed efficiency):the bottom quartile of sampled birds as measured by feed conversionratio. The taxonomic data from Example 26 were then compared to identifyspecies with a statically-significant difference relative abundancebetween the two cohorts. Specifically, for each OTU, the relativeabundance of the corresponding organism was compared between the twocohorts. Species were identified that exhibited a higher relativeabundance in low FCR with P<0.05 as determined by ANOVA. Cecalmicrobiota associated with improved feed efficiency were characterizedby an increased relative abundance of members of genera Parabacteroides,Akkermansia, Subdoligranulum, Bacteroides, Barnesiella (FIG. 18). Thespecies Bacteroides dorei and Barnesiella intestinihominis exhibited aparticularly strong associated with improved feed efficiency (FIG. 18).FIG. 18 provides box and scatter plots illustrating four taxa positivelyassociated with feed efficiency (P<0.05).

Example 28 Consistency of Taxa Associations and Putative Function

The Study of Example 25 was repeated multiple times to assess the impactof different regions, diet types, coccidiosis programs, bird genetics,and grow-out durations. The cecal microbiota obtained from samplingbirds and sequencing their gut microbiota according to the methods ofExamples 25-25 was repeated for the various studies. The contrastanalysis and correlation determinations of Example 27 were also repeatedfor the various studies, both independently and after pooling the datainto a single set.

Taxa that exhibited a consistent association with performance across themultiple studies were identified by performing a joint cohort analysis.Table 12 shows the taxa, representative members, and putative functionwere obtained.

TABLE 12 Cecal Microbiota Taxon Representative Members Putative FunctionTaxon 1 O. splanchnicus Increased body weight Taxon 2 B. clarusIncreased body weight Reduced feed conversion ratio Taxon 3 B. faecalis;B. dorei Reduced feed conversion ratio Taxon 4 B. intestinihominisReduced feed conversion ratio Taxon 5 Oscillibacter spp. Increased bodyweight

Furthermore, over 100 taxa were identified that exhibited either nocorrelation with growth performance or inconsistent association withgrowth performance between the various studies.

Example 29 Method of Enhancing Beneficial Taxa

The impact of the oligosaccharide preparations of Example 9 on theabundance of the beneficial taxa of Example 28 was assessed in vivo forcommercial broiler chickens. Specifically, the sampled birds obtainedfrom the study of Example 25 were grouped into two cohorts: (1) ControlGroup, including only those birds fed the control diets; and (2) TreatedGroup, including only those birds fed the treated diets containing theoligosaccharide preparation.

A contrast analysis was performed between Control Group and TreatedGroup cohorts. It was found that approximately twenty microbiome speciesexhibited a statistically significant shift in abundance as a result offeeding the oligosaccharide feed additive. Furthermore, it was foundthat multiple of the taxa associated with performance of Examples 27 and28 were increased as a result of feeding the animal the oligosaccharidepreparation. In particular: Taxon 4 (Barnesiella intestinihominis) wasincreased significantly (P<0.01, by ANOVA) and Taxon 1 (Odoribactersplanchnicus) was increased significantly (P<0.1, by ANOVA).Multiple-hypothesis testing confirmed that the beneficial taxa ofExample 29 were increased significantly in the Treatment Group cohort(Q<0.005, by Mann-Witney-Wilcoxon Testing).

Example 30 Method of Improving Growth Performance

Animals in the study of Example 25 fed treated diets exhibited astatistically significant increase in beneficial taxa and acorresponding statistically significant increase in growth performance,as measured by a treatment effect of 11 pts in body weight relative tocontrol (P<0.05).

Example 31 Upregulation of Endogenous Digestive Enzymes Expressed by theGut Microbiome

Ex vivo transcriptomics and an in vivo feed digestibility study wereperformed to demonstrate the ability of an oligosaccharide preparationto increase the transcription of genes known to code for enzymes thatcontribute to the digestion of feed and to demonstrate increased feeddigestibility in broiler chickens.

Ex Vivo Transcriptomics:

A microbiota sample obtained using the methods of Example 25 was used toprepare a 20% w/v suspension in pH 7.4 phosphate buffered saline (PBS)containing 15% glycerol. Working under anaerobic conditions, an aliquotof the suspension was centrifuged at 2,000 g, the supernatant wasremoved by pipette, and the pellet was resuspended to form a 1% w/vcecal slurry in a sterile aqueous mixture of: 900 mg/L sodium chloride,26 mg/L calcium chloride dihydrate, 20 mg/L magnesium chloridehexahydrate, 10 mg/L manganese chloride tetrahydrate, 40 mg/L ammoniumsulfate, 4 mg/L iron sulfate heptahydrate, 1 mg/L cobalt chloridehexahydrate, 300 mg/L potassium phosphate dibasic, 1.5 g/L sodiumphosphate dibasic, 5 g/L sodium bicarbonate, 0.125 mg/L biotin, 1 mg/Lpyridoxine, 1 m/L pantothenate, 75 mg/L histidine, 75 mg/L glycine, 75mg/L tryptophan, 150 mg/L arginine, 150 mg/L methionine, 150 mg/Lthreonine, 225 mg/L valine, 225 mg/L isoleucine, 300 mg/L leucine, 400mg/L cysteine, and 450 mg/L proline.

Deep-well microtiter plates (e.g., Costar 3958 plates) were loaded intriplicate with 250 uL of the above suspension, 50 uL of a sterile 20 wt% aqueous solution of selected oligosaccharide preparations from Example9, and 200 uL of sterile water. Control wells were loaded with 25 uL ofa sterile 20 wt % glucose solution and 200 uL of sterile water. Loadedplates were incubated at 37° C. for 45 hours under anaerobic conditionsfollowed by RNA extraction using methods known to one skilled in theart. The effect of the oligosaccharide preparation on the polysaccharideutilization loci (PUL) of the microbiota was determined by wholetranscriptome shotgun sequencing (RNA-seq). Relative to the glucosecontrol, increased transcription of was observed in the presence of theoligosaccharide preparation for multiple genes associated withhydrolytic digestion of feed: GH127 (CAZyme α-L-arabinofuranosidase),susC (outer membrane receptor associated with the starch utilizationsystem) and susD (starch binding protein). FIG. 19 illustrates increasedtranscription of hydrolytic digestive enzymes belonging to the PUL formicroflora fed the oligosaccharide preparation relative to control.

In Vivo Increase in Feed Digestibility

Ross 308 broilers were grown for 35 days in raised floor pens on aconventional three-phase diet program. Three dietary treatment groupswere compared: (1) a Negative Control Group fed an industryrepresentative wheat-soy poultry diet; (2) a Positive Control Group fedthe control diet supplemented with a conventional xylanase feed additive(AB-Vista Econase) at the recommended inclusion level; and (3) aTreatment Group fed the control diet supplemented with 500 ppm of anoligosaccharide preparation from Example 9. All diets contained ananticoccidial, industry standard levels of phytase and TiO₂ marker forperforming the digestibility determination. Conventional environmental,lighting, and monitoring programs were employed. A randomized blockstudy design was implemented with eight pens per treatment group withthirty-eight birds per pen.

Birds in the Treatment Group were observed to exhibit a statisticallysignificant 2.8% increase in dry matter digestibility and astatistically significant 4.5% increase in Nitrogen digestibilityrelative to the Negative Control Group. The Treatment Group alsooutperformed the Positive Control Group fed enzyme-supplemented diets.Positive control birds exhibited a 1.4% increase in dry matterdigestibility and a 1.5% increase in Nitrogen digestibility.

Example 32 Growth and Sampling Piglets Fed an OligosaccharidePreparation

Nursery pigs were grown on the diets of Example 15 to determine theeffect of an oligosaccharide preparation on the functional metagenomicsof the pig ileal, cecal, and fecal microbiomes.

One hundred and forty-four Redon x Large-White weaned piglets with aninitial body weight of 8.54±1.70 kg were raised for 42 days in flat-deckhousing in an environmentally controlled room. The animals wereallocated to 4 equal groups of 36 piglets in 9 pens of 4 animals perpen. Each pen had a plastic-coated welded wire floor and was equippedwith two water nipples and two stainless-steel individualized feeders.The room humidity was maintained at 50% and the temperature wasmaintained initially at 27° C. and lowered weekly by about 2° C. perweek to 21-22° C. The animals were fed according to a two-phase dietprogram formulated according to NRC (2012) nutritional recommendationsusing the diet constructions of Table 13.

TABLE 13 Nursery Pig Diet Compositions Ingredient Pre-Starter (wt %)Starter (wt %) Corn 52.20 62.25 Soy Bean Meal 26.00 26.00 Corn Starch6.00 — Soy Protein Concentrate 5.00 3.00 Calcium Carbonate 0.60 0.55Di-Calcium Phosphate 1.7 1.50 Sodium Bicarbonate 0.45 0.40 SodiumChloride 0.25 0.20 L-Lysine 0.80 0.65 L-Threonine 0.30 0.25 L-Methionine0.20 0.20 Soybean Oil 3.00 2.00 Vitamin & Mineral Premix 3.50 3.00

Pre-starter diets were fed from day 0 to day 14; and Starter Diets werefed from day 15 until day 42. All diets were provided ad libitum as mashdiet formulations. The pigs were grouped into treatment groups accordingto Table 14.

TABLE 14 Nursery Pig Treatment Groups Treatment Group Pre-Starter DietStarter Diet Control Diet 15.3(CTR) Example 15.5(CTR) Test Diet15.3(CTR) supplemented Diet 15.5(CTR) supplemented Group A with 125 ppmof with 125 ppm of oligosaccharide oligosaccharide preparation 9.7preparation 9.7 Test Diet 15.3(CTR) supplemented Diet 15.5(CTR)supplemented Group B with 250 ppm of with 250 ppm of oligosaccharideoligosaccharide preparation 9.7 preparation 9.7 Treatment Diet 15.3(TRT)Diet 15.6(TRT)

The average daily weight gain (ADWG) of piglets in the Treatment Groupwas determined to be 508.1 g/day compared to the Control Group whichgained 494.9 g/day. The feed conversion ratio for the Control Group wasfound to be 1.781 kg/kg, while for the Treatment Group it was found tobe 1.748.

Example 33 Effect of an Oligosaccharide Preparation on FunctionalMetagenomics

Nursery pigs were raised in pens according to the general protocol ofExample 32. The pigs were grouped into two treatment groups: (1) theControl Group was fed the diets of 15.3(CTR) and 15.5(CTR); and (2) theTreatment Group was fed the diets of 15.3(CTR) and 15.5(TRT). For eachtreatment group, 13 animals were selected at random, euthanized, anddissected to obtain ileal, cecal, and fecal (colon) microbiota samples.

The 69 microbiota samples were prepared by SAMBO DNA extraction andsequenced by MetaQuant (MGP). 21.6M cleaned reads were generated onaverage per sample. Mapping and counting were performed using METEOR.Mapping with Bowtie2 (identity 95%) was performed to remove hostcontaminants (pig genome), and the resulting sequences were profiledagainst a custom gene catalog containing 9M genes (ileal, cecal, andfecal microbiomes). Biostatistical analysis was performed usingMetaOMineR. Greater than 2×10⁷ high quality reads were obtained. Toavoid bias due to mapping rate variability, all samples were downsizedto 10M mapped reads and gene abundances were normalized by FPKMaccording to gene length and sequencing depth.

Gene Richness and Functional Metagenomic Analysis:

Samples were grouped into cohorts according to treatment group andmicrobiome type (ileal, cecal, fecal) and a contrast analysis wasperformed to determine statistically-significant changes in generichness, measured as the number of distinct genes. Animals in thetreatment group exhibited a 2-5-fold increase in gene richness,significant at P<0.006 for the cecal microbiota and P<0.06 for the fecalmicrobiota (Wilcoxon test). A numerical increase in the ileal generichness was also observed.

Genes were annotated using the KEGG database (v82) and grouped accordingto gut metabolic modules (GMMs) [Darzi, Y. et al., The ISME Journal, 10,1025-1028 (2015)] using an internal pipeline. A contrast analysis wasperformed on the GMM abundance data to determine metabolic pathways andfunctions that were modulated in the Treatment Group relative to theControl Group. FIG. 20 illustrates the effect of the oligosaccharidepreparation on the functional metagenomics of the piglet microbiome. Astatistically-significant increase in pathways associated withacetogenesis was observed in the ileum microflora.Statistically-significant increases in pathways associated with pectindegradation and SCFA production, among others, were observed in thececum. Furthermore, SCFA pathways for the production of butyrate andpropionate were enriched in the cecal microbiota for the TreatmentGroup.

Example 34 Reduction of Undesirable Microbial Species in the GutMicroflora

The impact of the oligosaccharide preparations of Example 9 onundesirable taxa was assessed in vivo for commercial broiler chickens.Specifically, the sampled birds obtained from the study of Example 25were grouped into two cohorts: (1) the Control Group, including onlythose birds fed the control diets; and (2) the Treated Group, includingonly those birds fed the treated diets containing the oligosaccharidepreparation.

A contrast analysis was performed between the Control Group and TreatedGroup cohorts to assess changes in the abundances of the taxa analyzedin Example 26. Birds fed the treated diet exhibited the followingchanges in microbial relative abundance as shown in Table 15.

TABLE 15 Relative Abundance of Microbial Species in Treated Group log₂(Fold Change from Microbial Species Control Group) Campylobacter jejuni−1.9 (P < 0.001) Helicobacter pullorum −1.3 (P < 0.001) Campylobactercoli −0.7 (P < 0.05)  Escherichia coli −0.7 (P < 0.1) 

FIG. 21 illustrates that the species in Table 15 are reduced in theTreatment Group relative to the Control Group. By contrast, 83 specieseither increased or did not decrease in relative abundance. Singlestrain growth experiments confirmed that the oligosaccharides were notantimicrobial.

Example 35 Consistency Across Multiple Studies

The Study of Example 25 was repeated multiple times to assess the impactof different regions, diet types, coccidiosis programs, bird genetics,and grow-out durations. The cecal microbiota obtained from sampled birdsin the additional studies were sequenced according to the methods ofExamples 25-26. The contrast analysis of Example 34 was also repeatedfor the various studies, both independently and after pooling the datainto a single set. A reduction in the Gram-Negative bacteria,Campylobacter spp., Heliobacteria spp., and Escherichia spp. wasobserved consistently across multiple studies. ProteobacteriaCampylobacter species was observed consistency in all of the studies, aswas an overall reduction in.

Example 36 Association Between Mortality and Intestinal PathogenAbundance

The effect of common pathogens in the intestinal microflora on animalhealth was assessed in broiler chickens.

Ross 708 straight run chicks were obtained from a common breeder flockand placed into twenty-four 8 ft×6 ft floor pens situated within asingle broiler house. 30 male chicks were placed per pen and birdweights by pen were recorded on Days 0. Birds were raised on built uplitter top-dressed with fresh wood shavings to provide a bedding with athickness of approximately 4 in. The temperature of the building wasmonitored and recorded daily. Environmental conditions during the trialfollowed industry practices. All birds were vaccinated following anindustry-standard vaccination program and were further vaccinated forcoccidiosis.

The birds were fed diets in two treatment groups according to afour-phase feeding program as shown in Table 16.

TABLE 16 Four Phase Feeding Program Diet Phase Feed Type Age (days)Starter crumble  0-16 Grower small pellet 16-28 Finisher pellet 28-42Withdrawal pellet 42-49

All diets were corn-soybean meal-based and contained dried distillersgrains with solubles (DDGS) and were free of antibiotic growth promotorsand ionophores or other anticoccidials. Birds in the Control Group (CTR)were fed the base diet, while birds in the Treatment Group (TRT) werefed the base diets supplemented with 500 ppm of an oligosaccharidepreparation from Example 9.

The diets were provided ad libitum via commercial Choretime automaticfeeders in each pen. From d 1 until 7, feed was also be supplied onfeeder trays, placed on the litter. Water was provided ad libitum fromone nipple drinking line. Standard floor pen management practices wereused throughout the experiment. Animals and housing facilities wereinspected twice daily, observing, and recording the general healthstatus, constant feed, and water supply as well as temperature, removingall dead birds, and recognizing unexpected events. Pen number andremoval date was recorded for all mortalities and culled birds over thecourse of the study.

On Day 15, one bird selected randomly from each pen was sampled forflood and cecal microbiota following the procedure of Example 25 and themicrobiota were analyzed according to the methods of Example 26. Thesampled birds were grouped into two cohorts: (1) High Mortality birdswere those drawn from pens in the top quartile for mortality; and (2)Low Mortality birds were those drawn from pens in the bottom quartilefor mortality. A contrast analysis was performed to determine microbialspecies with a statistically-significant difference in abundance betweenthe two cohorts. Approximately five species were found to correlatestrongly with mortality. Little or no correlation was found for morethan one hundred other cecal microbiota taxa. A particularly strongcorrelation was observed between the relative abundance of Escherichiaspecies and mortality. FIG. 22 provides a box and scatter plot forEscherichia coli (left panel) and other Escherichia species (rightpanel) clearly illustrating the connection between pathogen abundanceand bird mortality.

Example 37 Treatment Effect of Oligosaccharide Preparations

The sampled birds of Example 36 were analyzed by treatment group, forwhich a three-fold reduction was observed in the relative abundance ofEscherichia coli. Escherichia species were reduced overall (Q=0.07,multiple comparison by Mann-Whitney-Wilcoxon testing). Furthermore,sampled birds in the treatment group exhibited a three-fold reduction inthe number of pens exhibiting the apicomplexan parasite Eimeria tenella.

Example 38 Meta-Analysis of Live Growth Performance Studies in BroilerChickens

The effect of oligosaccharide preparations on the live growthperformance of commercial broiler chickens was assessed in vivo througha series of independent studies conducted across a variety of regions,times of year, background diet types, bird genetics, and managementpractices including litter handling and coccidiosis-control programs. Ineach study, birds were allotted to treatment groups including onecontrol group and one or more treated groups. The control group was fedonly the background diet. The treated groups were fed the backgrounddiet supplemented with a specified dose of the oligosaccharidepreparations of Example 9. In selected studies, a commercial feedadditive used in the poultry industry was included as a comparativeexample.

For each study, birds were housed in pens situated within a typicalbroiler house with a specified number (Hd/Rep) of birds in each pen.Statistical replications were implemented by randomly assigning pens totreatment groups, with a specified number (Reps/Trt) of replications pertreatment. Table 17 summarizes the protocol details for each studyincluded in the analysis.

TABLE 17 Protocol Cocci Study Country Season Length Diet Type Reps/TrtHd/Rep Genetics Sex Litter Program Ex. 38.1 USA Spring 35 Corn/Soy 6 14Cobb 500 M/F Used Saccox Ex. 38.2 USA Winter 49 Corn/Soy 12 60 Cobb 500M/F Used Maxiban Ex. 38.3 CA Winter 35 Corn/Soy 8 60 Ross 708 M UsedSaccox Ex. 38.4 USA Winter 49 Corn/Soy 12 60 Cobb 500 M/F Used MaxibanEx. 38.5 UK NA NA Wheat/Soy NA NA NA NA Clean None Ex. 38.6 USA Winter33 Corn/Soy 12 100  Cobb 500 M/F Used Amprol Ex. 38.7 USA Summer 49Corn/Soy 12 18 Hubbard M99 M Used None Ex. 38.8 C Winter 42 Corn/Soy 1217 Ross308 Male Used Vaccine Ex. 38.9 GB Autumn 42 Wheat/Soy 16 35Ross308 Male Fresh None Ex. 38.10 FR Autumn 42 Wheat/Soy 17 30 Ross308Male Fresh None Ex. 38.11 US Autumn 42 Corn/Soy 21 40 Cobb500 Male UsedVaccine Ex. 38.12 FR Summer 36 Corn/Soy 12 18 Cobb500 Male Fresh VaccineEx. 38.13 CA Spring 42 Corn/Soy 10 20 Ross708 Male Used Vaccine Ex.38.14 USA Spring 42 Corn/Soy 14 40 Cobb500 Male Used Vaccine Ex. 38.15NZ Spring 35 Wheat/Soy 12 20 Ross308 Male Fresh Vaccine

Study outcomes included bird weight (BW), feed intake (FI), feedconversion ratio (FCR), percent mortality (by head), and mortalityweight. Pen was the statistical unit. Where possible, spatial blockingwas implemented, and treatment groups were assigned randomly to blocks.

Background Diets:

Birds were fed in diet phases according to local industry practices fora total study length between 35 and 49 days. Starter phase diets weretypically provided as crumble from bird placement through study day 15.All diets were free of antibiotic growth promoters. Starter control dietconstructions are presented in Table 18 (NA=data not available fromsite).

TABLE 18 Start Control Diet Constructions % Corn % Wheat % Soy % CornCrude Crude AME Lysine Methionine Study Meal Meal Meal DDGS Protein Fat(kcal/kg) (SID) (SID) Ex. 38.1 63.5 NA 27.4 NA 22.1 NA 2988 1.35 NA Ex.38.2 0 NA 0 NA NA NA NA NA NA Ex. 38.3 63.4 NA 28.3 NA 20.9 NA 2940 1.14NA Ex. 38.4 NA NA NA NA NA NA NA NA NA Ex. 38.5 NA NA NA NA NA NA NA NANA Ex. 38.6 0 NA 0 NA NA NA 3011 NA NA Ex. 38.7 0 NA 0 NA NA NA NA NA NAEx. 38.8 54.52 0 34.38 5 22.23 2.81 2900 1.24 0.63 Ex. 38.9 0 51.78 30.50 21.31 5.74 2899  1.251  0.622 Ex. 38.10 0 55.1 28 0 22.49 5.42 2899 1.237 NA Ex. 38.11 58.353 2.377 29.992 5 20.3 NA 2900 1.33 NA Ex. 38.12NA NA NA NA 22 NA 3011 NA NA Ex. 38.13 56.08 0 34.1 5 22.23 2.31 29001.24 0.63 Ex. 38.14 58.353 0 29.992 5 20.3 NA 2900 1.33 NA Ex. 38.15 054.92 28.31 5 NA 6.9  2900 1.24 NA

Grower phase diets were provided as pellets from day 16 through day 24.Grower control diet constructions are presented in Table 19 (NA=data notavailable from site).

TABLE 19 Grower Phase Control Diet Constructions % Corn % Wheat % Soy %Corn Crude Crude AME Lysine Methionine Study Meal Meal Meal DDGS ProteinFat (kcal/kg) (SID) (SID) Ex. 38.1 68.6 NA 22 NA 19.95 NA 3059 1.2  NAEx. 38.2 NA NA NA NA NA NA NA NA NA Ex. 38.3 65.6 NA 26.3 NA 19.9 NA2988 1.06 NA Ex. 38.4 NA NA NA NA NA NA NA NA NA Ex. 38.5 NA NA NA NA NANA NA NA NA Ex. 38.6 NA NA NA NA NA NA 3102 NA NA Ex. 38.7 NA NA NA NANA NA NA NA NA Ex. 38.8 54.95 0 28.31 10 20.8 3.88 3000 1.11 0.56 Ex.38.9 0 57.135 26 0 19.01 6.55 2997 1.08  0.533 Ex. 38.10 0 55.77 24 020.94 7.4  2998 1.11 NA Ex. 38.11 65.383 0.344 20.404 10 17.5 NA 30401.33 NA Ex. 38.12 NA NA NA NA 19 NA 3035 NA NA Ex. 38.13 56.53 0 28.0210 20.8 3.39 3000 1.11 0.56 Ex. 38.14 65.383 0 20.404 5 17.5 NA 30401.14 NA Ex. 38.15 0 57.3 23.05 6 NA 6.27 3000 1.11 NA

Finisher phase diets were provided as pellets from day 16 through day24. Finisher control diet constructions are presented in Table 20(NA=data not available from site).

TABLE 20 Finisher Phase Control Diet Constructions % Corn % Wheat % Soy% Corn Crude Crude AME Lvsine Methionine Study Meal Meal Meal DDGSProtein Fat (kcal/kg) (SID) (SID) Ex. 38.1 74.3 NA 27.4 NA NA NA 31551.06 NA Ex. 38.2 NA NA NA NA NA NA NA NA NA Ex. 38.3 70.8 NA 28.3 NA NANA 3059 0.94 NA Ex. 38.4 NA NA NA NA NA NA NA NA NA Ex. 38.5 NA NA NA NANA NA NA NA NA Ex. 38.6 NA NA NA NA NA NA 3203 NA NA Ex. 38.7 NA NA NANA NA NA NA NA NA Ex. 38.8 57.37 0 24.85 10 19.4 5.16 3100 1.03 0.55 Ex.38.9 0 59.94 23 0 17.53 7.71 3097  1.003  0.503 Ex. 38.10 0 87.67 21 019.53 8.88 3099  0.994 NA Ex. 38.11 69.41 0.12 16.879 10 16 NA 3084 1.01NA Ex. 38.12 NA NA NA NA NA NA NA NA NA Ex. 38.13 58.95 0 24.56 10 19.44.66 3100 1.03 0.55 Ex. 38.14 69.41 0 16.879 5 16 NA 3084 1.01 NA Ex.38.15 0 62.02 17.62 6 NA 7.25 3100 0.99 NA

Treatment Groups:

For each study, the treatment groups were designed to compare the effectof oligosaccharide preparations versus the control diet. For selectedstudies, the treatment groups were designed to assess the dose-responsecurve for oligosaccharide preparations. Treated diets were obtained byblending a sufficient amount of the corresponding oligosaccharidepreparation from Example 9 such that the final oligosaccharide contentachieved the specified dose (units of ppm on a dry solids basis). Inselected studies, a comparative example (Comp. Ex 36) was provided by acommercial whole yeast product (Diamond V XPC Original). Treatments wereallocated according to Table 21.

TABLE 21 Treatment Group Allocations Treatment Treatment TreatmentTreatment Treatment Treatment Treatment Study Group 1 Group 2 Group 3Group 4 Group 5 Group 6 Group 7 Ex. 38.1 Control Ex. 9.7 (500 ppm) Ex.38.2 Control Ex. 9.7 (500 ppm) Ex. 38.3 Control Ex. 9.7 (500 ppm) Ex.38.4 Control Ex. 9.7 (500 ppm) Ex. 38.5 Control Ex. 9.7 Ex. 9.7 Comp.Ex. 36 (100 ppm) (500 ppm) (1500 ppm) Ex. 38.6 Control Ex. 9.7 (500 ppm)Ex. 38.7 Control Ex. 9.7 (500 ppm) Ex. 38.8 Control Ex. 9.7 Ex. 9.3 (500ppm) (500 ppm) Ex. 38.9 Control Ex. 9.7 (500 ppm) Ex. 38.10 Control Ex.9.7 Ex. 9.2 Ex. 9.3 Comp. Ex. 36 (500 ppm) (500 ppm) (500 ppm) (1250ppm) Ex. 38.11 Control Ex. 9.7 Ex. 9.2 Ex. 9.3 Ex. 9.4 Ex. 9.5 Comp. Ex.36 (500 ppm) (500 ppm) (500 ppm) (500 ppm) (500 ppm) (1250 ppm) Ex.38.12 Control Ex. 9.7 Ex. 9.3 (500 ppm) (500 ppm) Ex. 38.13 Control Ex.9.2 Ex. 9.2 Ex. 9.2 Ex. 9.2 Ex. 9.2 (100 ppm) (250 ppm) (500 ppm) (750ppm) (1000 ppm) Ex. 38.14 Control Ex. 9.3 Ex. 9.3 Ex. 9.3 Ex. 9.3 Ex.9.3 Ex. 9.7 (100 ppm) (250 ppm) (500 ppm) (750 ppm) (1000 ppm) (500 ppm)(continued, Ex. 9.2 Ex. 9.2 Ex. 9.2 Ex. 9.2 Ex. 9.2 Comp. Ex. 36 8-13)(100 ppm) (250 ppm) (500 ppm) (750 ppm) (1000 ppm) (1250 ppm) Ex. 38.15Control Ex. 9.2 Ex. 9.2 Ex. 9.2 Ex. 9.3 Ex. 9.3 Ex. 9.3 (100 ppm) (250ppm) (500 ppm) (100 ppm) (250 ppm) (500 ppm)

Standard equipment and methods known in the art were used to prepareboth the background and treated diets. For the treated diets,oligosaccharide preparations and comparative products were formulated ontop of the background diet and added to the mixer pre-pelleting.Oligosaccharide inclusion was confirmed by in-feed assaying.

Live Growth Phase and Sampling:

Feed and water were provided ad libitum. Commercial lighting andtemperature programs were implemented in each study according to localindustry practices for the corresponding region. Pens were inspecteddaily and the count and weight of any mortalities was recorded in thestudy log. No veterinary interventions were required.

For each diet phase, the total pen weight gain, the starting and endingnumber of birds, and the total feed consumption were measured for eachpen. For each pen, the average bird weight (BW) was calculated bydividing the total pen weight by the number of birds in the pen at thetime of weighing. For each pen, the feed conversion ratio (FCR) wascalculated by dividing the total feed intake over an interval by thetotal weight gain of the corresponding pen. FCRs were adjusted formortalities (FCRma) by adding back the total weight of mortalitiesduring the period. To account for differences in pen weight, FCRma wascorrected to a common body weight to obtain the corrected FCR (cFCR) foreach pen using methods known in the art. The correction factor wasdetermined for various bird genetics using published BW and FCRperformance objectives as a function of growth day for the correspondinggenetics.

In selected studies, one bird from each pen was selected randomly forsampling either on day 15 and/or on the final study day. For eachsampled bird, 5 mL of blood was drawn from a wing vein into serumvacutainers. After coagulation, serum was recovered by centrifugation,removed, and frozen on dry ice for later processing. Each sampled birdwas then euthanized according to local ethical procedures and dissected.Cecal contents were removed to 5 mL conical tubes and immediately flashfrozen for microbiome whole genome sequencing and cecal metabolomics. Asmall resection of ileal tissue was taken, treated to deactivate RNA andfrozen for later gene expression analysis.

Example 39 Study Meta-Analysis

A statistical meta-analysis of the in vivo studies of Example 38 wasperformed to assess the impact of oligosaccharide feed additives andcomparative products on bird performance versus birds fed control diets.The analysis employed a mixed linear model with treatment group as thefixed effect and random effects for study nested with block. Statisticalanalysis was performed in R version 3.4.4 (2018 Mar. 15). Outcomes wereassessed by least-squares means, with statistical significance atP<0.05. Pairwise comparisons were performed according to Tukey's methodand assigned alphabetical labels: a, b, c, d . . . . Treatments with nocommon letter in their Tukey grouping label differed significantly underpairwise comparison at P<0.05.

Feed Conversion Ratio:

Study effects for cFCR were significant at P<0.05. Oligosaccharidetreatments provided at least 2.7 pts improvement in cFCR at 500 ppminclusion over the control diet, versus the comparative example, whichprovided a 2.2 pts improvement in cFCR at 1250 ppm inclusion. Theoligosaccharide of Example 9.4 provided a 6.4 pt improvement in cFCR at500 ppm inclusion. The results of the meta-analysis for cFCR arepresented in Table 22.

TABLE 22 Meta-Analysis for cFCR Δ cFCR cFCR Tukey (pts vs TreatmentGroup (lsmean) SE df Grouping control) Control 1.6511 0.043 14 d Ex. 9.2(500 ppm) 1.6019 0.044 14 a −4.9 Ex. 9.3 (500 ppm) 1.6106 0.044 14 abc−4.1 Ex. 9.4 (500 ppm) 1.5876 0.044 14 a −6.4 Ex. 9.5 (500 ppm) 1.59480.044 14 ab −5.6 Ex. 9.7 (500 ppm) 1.6246 0.043 14 be −2.7 Comp. Ex. 36(1250 ppm) 1.6293 0.044 14 c −2.2

Oligosaccharide treatment groups exhibited a higher consistency ofeffect versus the comparative example, Comp. Ex. 38. For eacholigosaccharide included in multiple studies, the consistency of itseffect on cFCR was assessed by determining the fraction of studies inwhich a given value of cFCR improvement versus control was observed. Forexample, the oligosaccharide of Example 9.2 at 500 ppm inclusionprovided at least a 3 pt cFCR benefit in 80% of the studies, at least a4 pt cFCR benefit in 60% of the studies, at least a 5 pt cFCR benefit in40% of the studies, and at least a 6 pt cFCR benefit in 40% of thestudies. The comparative example at 1250 ppm inclusion provided a 3 ptcFCR benefit in only 25% of the studies and did not provide a 4 pt cFCRbenefit or higher in any of the studies. The results are presented inTable 23.

TABLE 23 Consistency of Treatment Effect on cFCR Treatment 1 pt 2 pt 3pt 4 pt 5 pt 6 pt Group benefit benefit benefit benefit benefit benefitEx. 9.2 100% 80% 80% 60% 40% 40% (500 ppm) Ex. 9.3 100% 83% 67% 67% 50%33% (500 ppm) Ex. 9.7  85% 85% 38% 23% 15%  0% (500 ppm) Comp. Ex. 36100% 100%  25%  0%  0%  0% (1250 ppm)

A clear dose response between cFCR and the inclusion rate ofoligosaccharides in the diet was observed. For the oligosaccharide ofEx. 9.2, a 2.4 pt cFCR benefit was observed at 100 ppm inclusion(P>0.05), a 3.7 pt cFCR benefit was observed at 250 ppm inclusion(P<0.05), an a 6.4 pt cFCR benefit was observed at 1000 ppm inclusion(P<0.05).

Bird Weight:

Study effects for BW were significant at P<0.05. Oligosaccharidetreatments provided at least 48.9 grams increased body weight over thecontrol diet at 500 ppm inclusion, versus the comparative example whichprovided 39.6 grams increased body weight at 1250 ppm inclusion. Theoligosaccharide of Example 9.5 provided 81.8 grams increased BW versuscontrol at 500 ppm inclusion. The results of the meta-analysis for BWare presented in Table 24.

TABLE 24 Meta-Analysis for BW Δ BW Treatment BW Tukey (g vs Group(lsmean) SE df Grouping control) Control 2,755 113 14 a 0 Ex. 9.2 (500ppm) 2,817 113 14 b 62.6 Ex. 9.3 (500 ppm) 2,807 113 14 b 52.6 Ex. 9.4(500 ppm) 2,838 114 14 b 83.3 Ex. 9.5 (500 ppm) 2,837 114 14 b 81.8 Ex.9.7 (500 ppm) 2,804 113 14 b 48.9 Comp Ex. 36 (1250 ppm) 2,794 113 14 b39.6

Flock Uniformity:

Birds fed oligosaccharide preparations at 500 ppm inclusion exhibitedimproved flock uniformity versus birds fed the control diet. For eachtreatment group, flock uniformity was assessed by calculating thefraction of bird weights that fell within a range of ±5% around the meanbird weight for its corresponding study. Averaged across all studies36.1-36.15, 81.7% of birds fed the control diet fell within ±5% of theaverage bird weight while 91.3% of birds fed diets treated with theoligosaccharide of Ex. 9.2 fell within ±5% of the average bird weight.The uniformity effect was significant at P<0.01 as measured by thenon-parametric Ansari-Bradley test.

Example 40 Microbiome Sequencing and Correlation Analysis

Cecal microbiome samples from Example 38 were processed and analyzed bywhole genome sequencing using methods comparable to those described inExample 26. The relative abundance of identified taxa was determined andcorrelated against bird performance parameters including feed efficiency(FCR) and weight (BW) by linear regression. As shown in Table 25, thefollowing taxonomic lineages provided the 94 strongest correlationsagainst performance endpoints.

TABLE 25 Taxonomic Lineages with 94 Strongest Correlations AgainstPerformance Endpoints BW FCR Taxonomic Lineage Correlation Correlationk_Bacteria; p_Lentisphaerae; c_Lentisphaeria; o_Victivallales;f_Victivallales_unclassified; 0.127342 −0.40176g_Victivallales_unclassified; s_Victivallales_bacterium_CCUG_44730k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; o_Enterobacterales;0.098983 −0.30691 f_Enterobacteriaceae; g_Escherichia;s_Escherichia_fergusonii k_Bacteria; p_Fusobacteria; c_Fusobacteriia;o_Fusobacteriales; f_Fusobacteriaceae; −0.03954 −0.27462g_Fusobactcrium; s_Fusobacterium_sp_CAG_439 k_Bacteria; p_Firmicutes;c_Negativicutes; o_Selenomonadales; f_Selenomonadaceae; 0.088303−0.24518 g_Megamonas; Unclassified k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Odoribacteraceae; −0.13797 −0.21085Unclassified; Unclassified k_Bacteria; p_Firmicutes; c_Erysipelotrichia;o_Erysipelotrichales; f_Erysipelotrichaceae; −0.07597 −0.20076Unclassified; Unclassified k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.06693 −0.19798g_Faecalibacterium; s_Faecalibacterium_prausnitzii k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Clostridiaceae; −0.20942−0.19739 g_Candidatus_Arthromitus;s_Candidatus_Arthromitus_sp_SFB_turkcy k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Lachnospiraceae; −0.15565 −0.19631g_Lachnoclostridium; s_Lachnoclostridium_sp_An14 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae;0.141503 −0.19581 g_Bacteroides; s_Bacteroides_intestinalis k_Bacteria;p_Finnicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; −0.07409−0.19479 g_Lachnoclostridium; s_Lachnoclostridium_sp_An_138 k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; 0.252334−0.18335 f_Desulfovibrionaceae; g_Bilophila; Unclassified k_Bacteria;p_Proteobacteria; c_Deltaproteobacteria; o_Desulfovibrionales; −0.17096−0.1824 f_Desulfovibrionaceae; g_Desulfovibrionaceae_unclassified;s_Desulfovibrionaceae_bacterium k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.11715 −0.18239 g_Anaerotruncus;s_Anaerotruncus_colihominis k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; 0.060701 −0.18053g_Anaeromassilibacillus; s_Anaeromassilibacillus_sp_An172 k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales; 0.079419−0.18011 f_Burkholdcriales_unclassified; g_Burkholderialcs_unclassified;s_Burkholderiales_bacterium_1_1_47 k_Bacteria; p_Firmicutes;c_Erysipelotrichia; o_Erysipelotrichales; f_Erysipelotrichaceae;0.196018 −0.17961 g_Faecalitalea; s_Faecalitalea_cylindroidesk_Bacteria; p_Firmicutes; Unclassified; Unclassified; Unclassified;Unclassified; Unclassified 0.110149 −0.16968 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae;0.245507 −0.15487 g_Bacteroides; s_Bacteroides_faecis k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; −0.25928−0.14818 g_Ruminococcaceae_unclassified; s_Clostridium_leptumk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;f_Odoribacteraceae; 0.147346 −0.14584 g_Butyricimonas;s_Butyricimonas_virosa k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Eubacteriaceae; 0.104361 −0.14004g_Eubacteriaceae_unclassified; s_Eubacteriaceae_bacterium_CHKCI005k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales; Unclassified;Unclassified; −0.1042 −0.13951 Unclassified k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; −0.12019 −0.12973g_Alistipes; s_Alistipes_onderdonkii k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; 0.091795 −0.12878g_Bacteroides; s_Bacteroides_thetaiotaomicron k_Bacteria;p_Actinobacteria; c_Actinobacteria; o_Bifidobacteriales;f_Bifidobacteriaceae; 0.166559 −0.12621 g_Bifidobacterium;s_Bifidobacterium_longum_CAG_69 k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; 0.236047 −0.12313g_Alistipes; s_Alistipes_sp_HGB5 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Lachnospiraceae; 0.155753 −0.12255 g_Anaerotignum;s_Anaerotignum_lactatifermentans k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; o_Enterobacterales; 0.233591 −0.12166f_Enterobacteriaceae; Unclassified; Unclassified k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; −0.14566−0.1155 g_Anaeromassilibacillus; Unclassified k_Bacteria;p_Proteobacteria; c_Betaproteobacteria; o_Burkholderiales;f_Sutterellaceae; 0.16901 −0.1151 g_Parasutterella;s_Parasutterella_excrementihominis k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; 0.230672 −0.10698g_Bacteroides; s_Bacteroides_vulgatus k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Rikenellaceae; 0.238234 −0.10519g_Alistipes; s_Alistipes_sp_CAG_29 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Lachnospiraceae; −0.20494 −0.10222g_Lachnoclostridium; s_Clostridium_saccharolyticum k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Lachnospiraceae; −0.12726−0.09614 g_Blautia; Unclassified k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Clostridiaceae; −0.21692 −0.09552 g_Clostridium;s_Clostridium_leptum_CAG_27 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Odoribacteraceae; −0.13067 −0.08206 g_Butyricimonas;s_Butyricimonas_sp_An62 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Eubacteriaceae; Unclassified; 0.25026 −0.06481Unclassified k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.235859 −0.04619 g_Alistipes;s_Alistipes_finegoldii k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.218292 0.055568 g_Alistipes;s_Alistipes_putredinis_CAG_67 k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; f_Lactobacillaccaci; g_Lactobacillus; 0.2285720.058781 s_Lactobacillus_salivarius k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; o_Enterobacterales; f_Enterobacteriaceae;−0.24378 0.063736 g_Shigella; Unclassified k_Bacteria; p_Firmicutes;c_Negativicutes; Unclassified; Unclassified; Unclassified; −0.242020.063797 Unclassified k_Bacteria; p_Lentisphaerae; c_Lentisphaeria;o_Victivallales; Unclassified; 0.197695 0.068584 Unclassified;Unclassified k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Lachnospiraceae; 0.167102 0.080242 Unclassified; Unclassifiedk_Bacteria; p_Proteobacteria; c_Epsilonproteobacteria;o_Campylobacterales; −0.288 0.080559 f_Helicobacteraceae;g_Helicobacter; s_Helicobacter_pullorum k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Ruminococcaccaceae; 0.313342 0.082109g_Subdoligranulum; s_Subdoligranulum_variabile k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae;0.163272 0.083306 g_Bacteroides; s_Bacteroides_massiliensis k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcacceae; 0.135390.083369 g_Faecalibacterium; Unclassified k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; 0.158109 0.084931g_Bacteroides; s_Bacteroides_uniformis k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 0.249112 0.089645g_Anaeromassilibacillus; s_Anaeromassilibacillus_sp_An250 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; f_Tannerellaceae;−0.11628 0.090798 g_Tannerella; s_Tannerella_sp_6_1_58FAA_CT1k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales;f_Lactobacillaceae; 0.247627 0.104938 g_Lactobacillus;s_Lactobacillus_johnsonii k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.105039 0.105814 g_Alistipes;s_Alistipes_sp_CAG_268 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.10307 0.107791 g_Flavonifractor;s_Flavonifractor_sp_An82 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.21527 0.108887 g_Alistipes;s_Alistipes_sp_CHKCI003 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Clostridiaceae; −0.19161 0.115591 g_Butyricicoccus;s_Butyricicoccus_pullicaecorum k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.16451 0.119402 g_Gemmiger;s_Gemmiger_sp_An50 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Eubactcriaceae; g_Eubacterium; −0.15551 0.130182s_Eubacterium_rectale_CAG_3_6 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.15675 0.130987 g_Flavonifractor;s_Flavonifractor_sp_(——)An91 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.10942 0.131152 g_Drancourtella;Unclassified k_Bacteria; p_Proteobacteria; Unclassified; Unclassified;Unclassified; Unclassified; −0.30972 0.132825 Unclassified k_Bacteria;p_Firmicutes; c_Erysipelotrichia; o_Erysipelotrichales;f_Erysipelotrichaceae; 0.203757 0.135764 g_Massiliomicrobiota;Unclassified k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales;f_Lactobacillaceae; 0.120135 0.142632 g_Lactobacillus;s_Lactobacillus_vaginalis k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Bacteroidaceae; −0.11504 0.146017 g_Bacteroides;s_Bacteroides_fragilis k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Clostridiaceae; 0.174945 0.151387 g_Clostridium;s_Clostridium_sp_ATCC_29733 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Bacteroidaceae; −0.06869 0.157261 g_Bacteroides;Unclassified k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.155621 0.15925 g_Alistipes;s_Alistipes_sp_An66 k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; f_Lactobacillaceae; 0.246012 0.162187g_Lactobacillus; s_Lactobacillus_reuteri k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; f_Bacteroidaceae; −0.07462 0.164746g_Bacteroides; s_Bacteroides_dorei k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Eubacteriaceae; 0.17506 0.165728g_Eubacterium; s_Eubacterium_sp_An11 k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Eubacteriaceae; 0.188604 0.167651g_Eubacterium; s_Eubacterium_sp_An3 k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; f_Lactobacillaceae; 0.158929 0.16852 g_Lactobacillus;s_Lactobacillus_helveticus k_Bacteria; p_Firmicutes;c_Firmicutes_unclassified; o_Firmicutes_unclassified; −0.17633 0.172219f_Firmicutes_unclassified; g_Firmicutes_unclassified; Unclassifiedk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Ruminococcaceae; −0.06748 0.177671 g_Flavonifractor;s_Flavonifractor_sp_(——)An_10 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; 0.118547 0.185369g_Pseudoflavonifractor; s_Pseudoflavonifractor_sp_An85 k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales;f_Clostridiales_unclassified; 0.067653 0.185637g_Clostridiales_unclassified; s_Clostridiales_bacterium_CHKCI001k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Lachnospiraceae; 0.161327 0.187129 g_Blautia; s_Blautia_sp_An81k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;f_Rikenellaceae; −0.10556 0.192622 g_Alistipes; Unclassified k_Bacteria;p_Firmicutes; c_Clostridia; o_Clostridiales; f_Ruminococcaceae; 0.2346530.206078 g_Pseudoflavonifractor; s_Pseudoflavonifractor_sp_An187k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;f_Rikenellaceae; 0.273404 0.213441 g_Alistipes; s_Alistipes_indistinctusk_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Lachnospiraceae; −0.0804 0.218662 g_Tyzzerella; s_TyzzerellaspAn114k_Bacteria; p_Firmicutes; c_Bacilli; o_Lactobacillales;f_Lactobacillaceae; 0.089093 0.2243 g_Lactobacillus;s_Lactobacillus_aviarius k_Bacteria; p_Firmicutes; c_Bacilli;o_Lactobacillales; f_Lactobacillaceae; 0.231413 0.225168g_Lactobacillus; s_Lactobacillus_ingluviei k_Bacteria; p_Firmicutes;c_Clostridia; o_Clostridiales; f_Eubacteriaceae; 0.105658 0.228612g_Eubacteriaceae_unclassified; s_Eubacteriaceae_bacterium_CHKCI004k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Eubacteriaceae; 0.085665 0.262875 g_Eubacterium; Unclassifiedk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;f_Rikenellaceae; 0.07405 0.266283 g_Alistipes; s_Alistipes_sp_An54k_Bacteria; p_Firmicutes; c_Clostridia; o_Clostridiales;f_Clostridiales_unclassified; 0.148222 0.269782g_Clostridiales_unclassified; s_Clostridiales_bacterium_CHKCI006k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;f_Odoribacteraceae; −0.11429 0.270529 g_Odoribacter;s_Odoribacter_sp_CAG_788 k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Ruminococcaceae; −0.14162 0.276379 g_Flavonifractor;s_Flavonifractor_plautii k_Bacteria; p_Firmicutes; c_Clostridia;o_Clostridiales; f_Clostridiaceae; 0.081329 0.278429 g_Clostridium;s_Clostridium_sp_CAG_678 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Rikenellaceae; 0.135766 0.279078 g_Alistipes;s_Alistipes_timonensis k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; f_Bacteroidaceae; 0.067539 0.298525 g_Bacteroides;s_Bacteroides_sp_D2 k_Bacteria; p_Firmicutes; c_Erysipelotrichia;o_Erysipelotrichales; f_Erysipelotrichaceae; 0.177296 0.318597g_Massiliomicrobiota; s_Massiliomicrobiota_sp_An80

Performance Grouping and SIMPER Analysis:

Taxa were assigned into four performance correlation groups: Group 1taxa were associated with improved FCR and improved BW; Group 2 taxawere associated with improved FCR but decreased BW; Group 3 taxa wereassociated with increased BW but poorer FCR; Group 4 taxa wereassociated with poorer FCR and deceased BW. Taxa assignments were madeby a performing Similarity Percentage (SIMPER) analysis (see forexample, Clarke, K. R. (1993). Non-parametric multivariate analyses ofchanges in community structure. Australian Journal of Ecology, 18(1),117-143.). A separate analysis was performed for young animals (Day 15samplings in Example 36) and later phase animals (Study Endpointsampling in Example 36). Significance was assessed by running 100 SIMPERpermutations.

SIMPER analysis of young birds (starter phase, day 15 of growth)identified the following taxonomic allocations to performancecorrelation groups (Table 26).

TABLE 26 Taxonomic Lineages with Performance Correlations PerformanceSIMPER Group Taxonomic Lineage Contribution Significance Group 1k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.023f_Rikenellaceae; g_Alistipes; s_Alistipes_sp_HGB5 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.020 P < 0.01f_Tannerellaceae; g_Parabacteroides; s_Parabacteroides_distasonisk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.014f_Tannerellaceae; g_Parabacteroides; s_Parabacteroides_merdae Group 2k_Bacteria; p_Proteobacteria; c_Gammaproteobacteria; 0.018o_Enterobacterales; f_Enterobacteriaceae; g_Escherichia;s_Escherichia_coli k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.014 f_Barnesiellaceae; g_Barnesiella;s_Barnesiella_intestinihominis k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 0.030 P < 0.01 o_Enterobacterales;f_Enterobacteriaceae; g_Escherichia; s_Escherichia_coli k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.025f_Barnesiellaceae; g_Barnesiella; s_Barnesiella_intestinihominis Group 3k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.093f_Rikenellaceae; g_Alistipes; s_Alistipes_sp_CHKCI003 k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.020 f_Bacteroidaceae;g_Bacteroides; s_Bacteroides_xylanisolvens k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.108 f_Rikenellaceae; g_Alistipes;s_Alistipes_sp_CHKCI003 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.016 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_xylanisolvens k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.015 P < 0.05 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_uniformis Group 4 k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.085 f_Barnesiellaceae; g_Barnesiella;s_Barnesiella_sp_An22 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.075 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_fragilis k_Bacteria; p_Proteobacteria;c_Epsilonproteobacteria; 0.043 o_Campylobacterales; f_Helicobacteraceae;g_Helicobacter; s_Helicobacter_pullorum k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 0.013 o_Enterobacterales; f_Enterobacteriaceae;g_Shigella; s_Shigella_sonnei k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.089 f_Barnesiellaceae; g_Barnesiella;s_Barnesiella_sp_An22 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.071 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_fragilis k_Bacteria; p_Proteobacteria;c_Epsilonproteobacteria; 0.036 o_Campylobacterales; f_Helicobacteraceae;g_Helicobacter; s_Helicobacter_pullorum k_Bacteria; p_Proteobacteria;c_Gammaproteobacteria; 0.022 P < 0.05 o_Enterobacterales;f_Enterobacteriaceae; g_Shigella; s_Shigella_sonnei

SIMPER analysis of birds at the end of their grow-out identified thefollowing taxonomic assignments to performance correlation groups ispresented in Table 27.

TABLE 27 Taxonomic Assignments to Performance Correlation GroupsCorrelation SIMPER Group Taxonomic Lineage Contribution SignificanceGroup 1 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;0.058 P < 0.01 f_Tannerellaceae; g_Parabacteroides;s_Parabacteroides_distasonis k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.020 f_Tannerellaceae; g_Parabacteroides;s_Parabacteroides_merdae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.013 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_vulgatus k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.051 f_Tannerellaceae; g_Parabacteroides;s_Parabacteroides_distasonis k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.012 f_Tannerellaceae; g_Parabacteroides;s_Parabacteroides_merdae k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.009 f_Rikenellaceae; g_Alistipes; s_Alistipes_sp_HGB5Group 2 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales;0.015 f_Barnesiellaceae; g_Barnesiella; s_Barnesiella_intestinihominisk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.041f_Barnesiellaceae; g_Barnesiellai; s_Barnesiella_intestinihominisk_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.011 P <0.05 f_Odoribacteraceae; g_Butyricimonas; s_Butyricimonas_sp_An62 Group3 k_Bacteria; p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.044f_Rikenellaceae; g_Alistipes; s_Alistipes_sp_CHKCI003 k_Bacteria;p_Verrucomicrobia; c_Verrucomicrobiae; 0.022 o_Venucomicrobiales;f_Akkermansiaceae; g_Akkermansia; s_Akkermansia_muciniphila k_Bacteria;p_Bacteroidetes; c_Bacteroidia; o_Bacteroidales; 0.011 f_Rikenellaceae;g_Alistipes; s_Alistipes_sp_An54 k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.048 f_Rikenellaceae; g_Alistipes;s_Alistipes_sp_CHKCI003 k_Bacteria; p_Verrucomicrobia;c_Verrucomicrobiae; 0.020 o_Venucomicrobiales; f_Akkermansiaceae;g_Akkermansia; s_Akkermansia_muciniphila k_Bacteria; p_Firmicutes;c_Firmicutes_unclassified; 0.014 P < 0.01 o_Firmicutes_unclassified;f_Firmicutes unclassified; g_Firmicutes_unclassified;s_Firmicutes_bacterium_CAG_94 k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.012 f_Rikenellaceae; g_Alistipes;s_Alistipessp_An54 Group 4 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.124 f_Barnesicllaceae; g_Barnesiella;s_Barnesiellasp_An22 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.075 P < 0.01 f_Odoribacteraceae; g_Odoribacter;s_Odoribacter_sp_CAG_788 k_Bacteria; p_Proteobacteria;c_Epsilonproteobacteria; 0.067 o_Campylobacterales; f_Helicobacteraceae;g_Helicobacter; s_Helicobacter_pullorum k_Bacteria;p_Bacteroidctcsic_Bacteroidia; o_Bacteroidales; 0.016 f_Bacteroidaceae;g_Bacteroides; s_Bacteroides_dorei k_Bacteria; p_Bactcroidetes;c_Bacteroidia; o_Bacteroidales; 0.016 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_clarus k_Bacteria; p_Bactcroidctcsi; c_Bacteroidia;o_Bacteroidales; 0.113 f_Barnesiellaceae; g_Barnesiella;s_Barnesiella_sp_An22 k_Bacteria; p_Proteobacteria;c_Epsilonproteobacteria; 0.070 o_Campylobacterales; f_Helicobacteraceae;g_Helicobacter; s_Helicobacter_pullorum k_Bacteria; p_Bacteroidetes;c_Bacteroidia; o_Bacteroidales; 0.067 f_Odoribacteraceae; g_Odoribacter;s_Odoribacter_sp_CAG_788 k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.015 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_clarus k_Bacteria; p_Bacteroidetes; c_Bacteroidia;o_Bacteroidales; 0.012 f_Bacteroidaceae; g_Bacteroides;s_Bacteroides_dorei

It was observed that different oligosaccharide treatments resulted indistinct shifts in abundance between the various Performance CorrelationGroups.

Example 41 Method of Modulating Taxonomic Performance Groups

Birds are fed a diet comprising an oligosaccharide preparation ofExample 9, where the oligosaccharide identity is selected to increasethe abundance of taxa in Group 1, Group 2 and/or Group 3 of Example 40.

Birds are fed diets comprising an oligosaccharide preparation of Example9, where the oligosaccharide identity is selected to decrease theabundance of taxa in Group 4 of Example 40.

Example 42 Replicate Batches Scale-Up for Manufacturing

The production scale of oligosaccharide preparations was increased tothat of a 720 L overhead-stirred tank reactor. Twelve batch reactionsusing a scaled-up procedure derived from those of Example 9.2 wereperformed at the 720 L scale. The resulting oligosaccharide preparationswere characterized against pre-determined QC acceptance criteria toperform batch qualification and to assess the process stability.

For the twelve batches, process conditions such as temperature, reactiontime, and reaction pressure were varied intentionally in a range aroundthe nominal conditions of Example 9.2 to assess the sensitivities of theresulting product to reasonable variations in the process conditionsthat might be expected in a typical manufacturing environment. Forselect batches, an in situ viscosity probe was used to monitor the timedependence of the viscosity of the reactor contents. In certain batches,the reaction stopping time employed an in-process control (IPC) based onthe continuous viscosity measurement. Material amounts, including thedispensed quantities of reactants, distillation water, and evolvedcondensate were measured either by mass via load cells on the reactorand auxiliary tanks or volumetric flow and time.

The final water content of the reactor product was measured by KarlFisher titration for a representative aliquot of the reactor contentsdrawn at the end of the reaction, i.e., prior to pH neutralization anddilution. At a reaction temperature of 120° C., the water content of thereaction product was determined to be 8 and 9 wt % water on an as-isbasis. At a reaction temperature of 130° C., the water content of thereaction product was determined to be between 5 and 7 wt % water on anas-is basis.

The resulting oligosaccharide syrup appearance of all the batches wasdetermined by visual inspection as a caramel syrup. The total dissolvedsolids content was determined by Karl-Fisher titration, the residualmonomer content, MWn and MWw were determined by HPLC/GPC chromatography,the pH was determined by calibrated pH meter and the anhydro-DP2 contentwas determined by LC-MS/MS. As shown in Table 29, the following batchcharacterization data were obtained (N/R=“data not reported”):

TABLE 29 Characterization of Oligosaccharide Preparations Anhydro DP2Content (g wt % Residual wt % Anhydro DP2/ Batch DS pH Catalyst DPI MWnMWw g total DP2) 27.1 66.4 N/D N/R 17.5 777 1218 0.84% 27.2 68.8 3.3 N/R17.9 735 1091 0.91% 27.3 69.4 3.1 0.095 14.8 807 1276 N/R 27.4 71.0 N/D0.068 15.5 793 1241 1.04% 27.5 70.9 3.2 0.057 15.8 777 1196 1.15% 27.670.9 3.3 N/R 16.3 773 1170 N/R 27.7 70.7 3.0 N/R 15.7 783 1226 1.13%27.8 70.5 3.9 N/R 16.1 785 1182 1.09% 27.9 71.1 4.1 N/R 17.1 761 11691.09% 27.10 70.4 4.1 N/R 16.3 778 1193 1.15% 27.11 70.5 4.7 N/R 18.6 696995 1.33% 27.12 70.9 3.9 N/R 16.7 769 1194 1.12%

Example 43 pH Adjustment of an Oligosaccharide Preparation

The pH of the oligosaccharide preparation of Example 9.2 at 50 wt %solids content was determined in triplicate by diluting 5.00±0.05-gramaliquots of the oligosaccharide preparation with 1.80±0.02 mL ofdeionized water and mixing by vortex agitation to obtain a uniformconcentration. The pH of each aliquot was measured with a calibrated pHmeter (VWR, Symphony B30PCI) to obtain an average reading of 2.4 pHunits.

To 1.2 kg of the oligosaccharide preparation of Example 9.2 was added6.53 mL of 1.0 molar aqueous sodium hydroxide solution. The resultingmixture was mixed vigorously to achieve a uniform pH-adjusted syrup. ThepH of the resulting adjusted syrup at 50 wt % solids content was thendetermined in triplicate as described above to obtain an average of 4.1pH units.

The pH adjustment procedure was repeated for replicate batch synthesesat various scales, but with certain variations in the procedure by whichthe base was provided to the product oligosaccharide composition. Forone batch, the pH adjustment was performed as the final step of thereaction, prior to the dilution of reaction water. In another batch, thepH adjustment was performed concurrently with the dilution step by firstdissolving the required amount of base in the dilution water; the baseand dilution water were therefore added together to quench the reactiona single step to produce a final syrup at the desired pH. In anotherbatch, the base was provided as food-grade sodium hydroxide pellets. Inanother batch, 10 ppm of a food-grade silicone emulsion (Dow XiameterAFE-0100) was added to the reaction prior to dilution and pH adjustment.

Example 44 Preparation of a Glass Powder Formulation of anOligosaccharide Preparation

Approximately 50 grams of the oligosaccharide preparation of Example 9.1was dispensed onto a drying tray and placed in a forced-air convectionheater at 60° C. to produce a caramel colored brittle glass. The glasswas removed from the drying tray and ground with a shear rotary mill toyield a light-orange colored flowable powder. The particle size of thepowder was determined by sieving to be between 100 and 2000 microns,with 90% of the mass below 1350 microns. The true density of the coarsemilled powder was determined by Helium Pyncnometry to be 1.3063 g/mL.The resulting powder was observed to be flowable.

The formulation procedure was repeated using a hammer mill to obtain afine powder with 90% of the mass of the powder exhibiting a particlesize below 196 microns. The true density of the fine milled powder wasdetermined to be 1.5263 g/mL. The resulting powder was neither stablenor flowable.

DSC measurements were performed on the powders using two temperaturecycling programs. In the first program, temperature was ramped to 160°C. from 0° C. at a rate of 5° C./min, then annealed back to 0° C. at arate of −5° C./min, followed by a final heating back to 160° C. In thesecond program, the temperature was ramped to 50° C. from −50° C. at arate of 5° C./min, annealed to −60° C. at a rate of −5° C./min and thenheated to 60° C. at a rate of 5° C./min. The powder was observed toexhibit a glass transition temperature of between 20 and 40° C.,dependent on the residual water content of the solid between 5 and 10 wt% moisture.

The milling formulation process was repeated for each of theoligosaccharide preparations of Example 9.2, Example 9.3, Example 9.4,and Example 9.5. The powders readily re-dissolved in water andalcohol-water mixtures, but were insoluble in acetone, methanol, andanhydrous ethanol.

Example 45 Preparation of a Carrier-Loaded Powder Formulation

Equal masses of a 70 wt % aqueous syrup of the oligosaccharidepreparation of Example 9.2 and diatomaceous earth were combined at roomtemperature to yield a stable, flowable powder. The resulting powdercomprised about 35 wt % adsorbed oligosaccharide (dry solids basis) andabout 50 wt % carrier. The particle size distribution of the powder wasmeasured by sieving. 10% by weight of the powder exhibited a particlesize below 290 micrometers, 50% by weight of the powder exhibited aparticle size below 511 micrometers, and 90% by weight of the powderexhibited a particle size below 886 micrometers. The powder was stableto segregation and cohesion, as determined using standard aeration andcompressibility tests. The true density of the resulting powder wasmeasured by Helium pyncnometry to be 1.8541 g/mL.

The carrier loading formulation was repeated using feed-grade silica toyield a stable, flowable powder with a loading of at least 50 wt %oligosaccharide preparation (dry solids basis) with respect to the finalpowder. The true density of the resulting powder was measured to be1.5562 g/mL.

Example 46 Preparation of an Extruded Solid Form

A solid extruded product was prepared by blending 20% of theoligosaccharide preparation of Example 9.2 with semolina and formulatedthe mixture through a jacketed twin-screw dye extruder to form aflowable powder with a particle size between 0.2 mm and 3.0 mm, with 90%of the mass below 2 mm particle size. The resulting powder was observedto be free-flowing and stable.

Example 47 Preparation of Stable Powder Formulations

The solid formulations, including those of Examples 44-46, were assessedto determine their stability and hygroscopicity. The powders of Examples45 and 46 were observed to be stable to segregation and agglomeration,while the coarse milled powder of Example 44 was observed to be unstablewith respect to segregation.

Sample of each powder formulation to be tested were placed in a sealedclimate chambers at 50% relative humidity and 65% relative humidity forup to two-weeks exposure at 25° C. Of the forms tested, severalexhibited little or no mass gain upon exposure to humidity and remainedflowable after the two-week exposure period. The fine-milled powder ofExample 45 was found to be unstable with exposure to humidity.

Example 48 Determination of Residual Catalyst in OligosaccharidePreparations

The residual acid catalyst content of oligosaccharides preparations wasdetermined by Ion Chromatography. Between 80 and 100 milligrams of apowder formulation of the oligosaccharide preparation (obtained forexample as described in Example 44) were dissolved in exactly 1.00milliliter and centrifuged to remove particulates if necessary. Theresulting solution was analyzed by ion chromatography at 30° C. using aThermo Dionex ICS-3000 System equipped with conductivity detection, a4×250 mm Ion Pac AS19A column, an Ion Pac AS19G 50 4×50 mm pre-columnand a continuously regenerated CR-ATC anion trap column using KOH inwater as the eluent. Elution was conducted at 10 mM KOH for the firstten minutes after injection followed by a gradient elution increasinglinearly to 55 mM KOH at 25 minutes, then decreasing to 10 mM KOH at 26minutes, and remaining at 10 mM KOH until the end of the program.

For the oligosaccharide preparation of Example 9.2, the concentration ofresidual catalyst was determined by reference to a standard calibrationcurve generated using an authentic sample of (+)-camphor-10-sulfonicacid. A representative batch of the oligosaccharide preparation ofExample 9.2 was analyzed and the residual catalyst concentration wasdetermined to be 0.62 mg per gram of 70 wt % syrup.

Example 49 Qualification of the Residual Catalyst Concentration forBatch Acceptance

The residual catalyst determination of Example 48 was compared against abatch acceptance criterion to determine suitability of the batch forfurther use. The acceptance limit for the concentration of residualcatalyst in the product oligosaccharide preparation was preestablishedto be <1.0 mg per gram product syrup. The measured value of the residualcatalyst was 0.62 mg per gram of product syrup. Therefore, theacceptance criterion was met for the tested batch and the batch wasaccepted for further use.

Example 50 Formulation of a Syrup Product

The oligosaccharide preparation of Example 9.7 was pH adjusted to a pHof 4.2 with food grade sodium hydroxide according to the procedure ofExample 43. The resulting syrup was packaged into a 20 liter carboy witha tamper-resistant cap. Immediately prior to sealing the container, a500 gram sample was taken and subjected to quality testing. The totalsolids content of the syrup was confirmed to be greater than 70 wt %,per the methods of FCC APPENDIX X: Carbohydrates (Starches, Sugars, andRelated Substances): TOTAL SOLIDS. The reducing sugar content wasconfirmed to be less than 50% as D-glucose on a dry weight basisaccording to the method of FCC APPENDIX X: Carbohydrates (Starches,Sugars, and Related Substances): REDUCING SUGARS ASSAY. Sulfated ash wasconfirmed to be less than 1% on a dry weight basis using the method ofFCC APPENDIX II: Physical Tests and Determinations: C. OTHERS: RESIDUEON IGNITION (Sulfated Ash) Method II (for Liquids). The sulfur dioxidecontent was confirmed below 40 mg/kg using an optimized Monier Williamsmethod. The lead content was confirmed to be below 1 mg/kg using themethod of AOAC International Official Method 2013.06. The total aerobicplate count was confirmed to be below 1000 cfu/g using the methods ofCMMEF Chapter 7. Total yeast and mold were confirmed below 100 cfu/gusing the method of AACC International Approved Method 42-50. Coliformswere confirmed below 10 MPN/g using the method of the FDA BAM Chapter 4.E. coli was confirmed below 3 MPN/g using the method of FDA BAM Chapter4. Salmonella was confirmed to be not detected per a 25 gram sampleaccording to the method of FDA BAM Chapter 5. Staphylococcus aureus wasconfirmed to be below 10 cfu/g using the method of FDA BAM Chapter 12.Color was confirmed by visual inspection to be caramel. The containerwas sealed, the remaining retention sample was frozen and stored forfuture reference, and a certificate of analysis was issued for theresulting lot.

Example 51 Preparation of Treated Drinking Water

Drinking water containing 250 ppm of the oligosaccharide preparation ofExample 9.7 was prepared as follows. 37 mL of the oligosaccharide syrupof Example 50 and 40 grams of potassium sorbate were added gradually to50 gallons of potable tap water in a 55 gallon blue-poly drum. Thesolution was mixed manually using a paddle for 10 minutes at roomtemperature.

The method was repeated without the incorporation of potassium sorbate.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. It is not intendedthat the invention be limited by the specific examples provided withinthe specification. While the invention has been described with referenceto the aforementioned specification, the descriptions and illustrationsof the embodiments herein are not meant to be construed in a limitingsense. Numerous variations, changes, and substitutions will now occur tothose skilled in the art without departing from the invention.Furthermore, it shall be understood that all aspects of the inventionare not limited to the specific depictions, configurations or relativeproportions set forth herein which depend upon a variety of conditionsand variables. It should be understood that various alternatives to theembodiments of the invention described herein can be employed inpracticing the invention. It is therefore contemplated that theinvention shall also cover any such alternatives, modifications,variations or equivalents.

1. A method of increasing the body weight of an animal, the methodcomprising: administering a nutritional composition comprising a basenutritional composition and a synthetic oligosaccharide preparation toan animal, wherein said synthetic oligosaccharide preparation comprisesat least n fractions of oligosaccharides each having a distinct degreeof polymerization selected from 1 to n (DP1 to DPn fractions), wherein nis an integer greater than 3; and wherein each of a DP1 and DP2 fractionindependently comprises from about 0.5% to about 15% of anhydro-subunitcontaining oligosaccharides by relative abundance as determined by massspectrometry, and wherein the body weight of said animal is increasedrelative to the body weight of said animal prior to said administeringsaid nutritional composition to said animal, and wherein the increase inthe body weight of said animal is a larger increase relative to anincrease in body weight of a comparable control animal administered acomparable nutritional composition lacking said oligosaccharidepreparation.
 2. (canceled)
 3. The method of claim 1, wherein said bodyweight of said animal is at least at least 10 g, 20 g, 30 g, 40 g, 50 g,60 g, 70 g, 80 g, 90 g, or 100 g higher than said body weight of saidanimal prior to administration of said nutritional compositioncomprising said synthetic oligosaccharide preparation, as measured afterat least 30 days, 35 days, 40 days, 45 days, 50 days, 60 days, 70 days,80 days, or 90 days after first administration of said nutritionalcomposition comprising said synthetic oligosaccharide preparation, andwherein said animal ingests said nutritional composition at least onceduring every twenty-four-hour period.
 4. A method of decreasing the feedconversion ratio of an animal, the method comprising: administering anutritional composition comprising a base nutritional composition and asynthetic oligosaccharide preparation to an animal, wherein saidsynthetic oligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein the feed conversion ratio (FCR) of said animalis decreased relative to the FCR of said animal prior to saidadministering said nutritional composition to said animal.
 5. (canceled)6. The method of claim 4, wherein said feed conversion ratio (FCR) ofsaid animal is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% lowerthan said FCR of said animal prior to administration of said nutritionalcomposition comprising said synthetic oligosaccharide preparation, asmeasured after at least 30 days, 35 days, 40 days, 45 days, 50 days, 60days, 70 days, 80 days, or 90 days after first administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation, and wherein said animal ingests said nutritionalcomposition at least once during every twenty-four-hour period. 7.(canceled)
 8. A method of increasing the feed efficacy of an animal, themethod comprising: administering a nutritional composition comprising abase nutritional composition and a synthetic oligosaccharide preparationto an animal, wherein said synthetic oligosaccharide preparationcomprises at least n fractions of oligosaccharides each having adistinct degree of polymerization selected from 1 to n (DP1 to DPnfractions), wherein n is an integer greater than 3; and wherein each ofa DP1 and DP2 fraction independently comprises from about 0.5% to about15% of anhydro-subunit containing oligosaccharides by relative abundanceas determined by mass spectrometry, and wherein the feed efficiency ofsaid animal is increased relative to the feed efficiency of said animalprior to said administering said nutritional composition to said animal.9. The method of claim 8, wherein said feed efficiency of said animal isat least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% higher than saidfeed efficiency of said animal prior to administration of saidnutritional composition comprising said synthetic oligosaccharidepreparation.
 10. The method of claim 8 or 9, wherein said feedefficiency of said animal is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, or 10% higher than said feed efficiency of said animal prior toadministration of said nutritional composition comprising said syntheticoligosaccharide preparation, as measured after at least 30 days, 35days, 40 days, 45 days, 50 days, 60 days, 70 days, 80 days, or 90 daysafter first administration of said nutritional composition comprisingsaid synthetic oligosaccharide preparation, and wherein said animalingests said nutritional composition at least once during everytwenty-four-hour period. 11-15. (canceled)
 16. The method of claim 9,wherein said nutritional composition comprises at least 100 ppm, 200ppm, 300 ppm, 400 ppm, 500 ppm, 600 ppm, 700 ppm, 800 ppm, 900 ppm, 1000ppm, 1500 ppm, or 2000 ppm oligosaccharide preparation. 17-20.(canceled)
 21. A method of modulating the growth of at least onemicrobial species in the gastrointestinal tract of an animal, saidmethod comprising: administering a nutritional composition comprising abase nutritional composition and a synthetic oligosaccharide preparationto an animal, wherein said synthetic oligosaccharide preparationcomprises at least n fractions of oligosaccharides each having adistinct degree of polymerization selected from 1 to n (DP1 to DPnfractions), wherein n is an integer greater than 3; and wherein each ofa DP1 and DP2 fraction independently comprises from about 0.5% to about15% of anhydro-subunit containing oligosaccharides by relative abundanceas determined by mass spectrometry, and wherein a level of at least onemicrobial species in a gastrointestinal sample from said animal isincreased or decreased relative to a level of said at least onemicrobial species in a gastrointestinal sample from said animal prior tosaid administering said nutritional composition to said animal. 22.(canceled)
 23. A method of modulating the growth of at least onemicrobial species in the gastrointestinal tract of an animal, saidmethod comprising: administering a nutritional composition comprising abase nutritional composition and a synthetic oligosaccharide preparationto an animal, wherein said synthetic oligosaccharide preparationcomprises at least n fractions of oligosaccharides each having adistinct degree of polymerization selected from 1 to n (DP1 to DPnfractions), wherein n is an integer greater than 3; and wherein each ofa DP1 and DP2 fraction independently comprises from about 0.5% to about15% of anhydro-subunit containing oligosaccharides by relative abundanceas determined by mass spectrometry, and wherein a level of at least onemicrobial species in a gastrointestinal sample from said animal isincreased or decreased relative to a level of said at least onemicrobial species in a gastrointestinal sample from a comparable controlanimal that has been administered a comparable nutritional compositionlacking said synthetic oligosaccharide preparation.
 24. The method ofclaim 21 or claim 23, wherein said at least one microbial species is anarchaea, a bacteria, a protozoan, a virus, a bacteriophage, a parasite,or a fungus.
 25. The method of claim 23, wherein said at least onemicrobial species is an archaea, a bacteria, a protozoan, a virus, abacteriophage, a parasite, or a fungus. 26-34. (canceled)
 35. The methodof claim 24, wherein said method comprises promoting the growth of saidat least one microbial species, and wherein said level of at least onemicrobial species in said gastrointestinal sample is increased relativeto a level of said at least one microbial species in a gastrointestinalsample from a comparable control animal that has been administered acomparable nutritional composition lacking said syntheticoligosaccharide preparation. 36-100. (canceled)
 101. A method ofmodulating expression of at least one microbial protein within thegastrointestinal tract of an animal, said method comprising:administering a nutritional composition comprising a base nutritionalcomposition and a synthetic oligosaccharide preparation to an animal,wherein said synthetic oligosaccharide preparation comprises at least nfractions of oligosaccharides each having a distinct degree ofpolymerization selected from 1 to n (DP1 to DPn fractions), wherein n isan integer greater than 3; and wherein each of a DP1 and DP2 fractionindependently comprises from about 0.5% to about 15% of anhydro-subunitcontaining oligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial protein in agastrointestinal sample is increased or decreased relative to a level ofsaid at least one microbial protein in a gastrointestinal sample fromsaid animal prior to administration of said nutritional composition tosaid animal.
 102. (canceled)
 103. A method of modulating expression ofat least one microbial protein within the gastrointestinal tract of ananimal, the method comprising: administering a nutritional compositioncomprising a base nutritional composition and a syntheticoligosaccharide preparation to an animal, wherein said syntheticoligosaccharide preparation comprises at least n fractions ofoligosaccharides each having a distinct degree of polymerizationselected from 1 to n (DP1 to DPn fractions), wherein n is an integergreater than 3; and wherein each of a DP1 and DP2 fraction independentlycomprises from about 0.5% to about 15% of anhydro-subunit containingoligosaccharides by relative abundance as determined by massspectrometry, and wherein a level of at least one microbial protein in agastrointestinal sample is increased or decreased relative to a level ofsaid at least one microbial protein in a gastrointestinal sample from acomparable control animal that has been administered a comparablenutritional composition lacking said synthetic oligosaccharidepreparation.
 104. The method of claim 101 or claim 103, wherein saidlevel of said at least one microbial protein in said gastrointestinalsample is decreased relative to said level of said at least onemicrobial protein in a gastrointestinal sample from said animal prior toadministration of said nutritional composition to said animal. 105-188.(canceled)